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Heme-based oxygen sensor protein FixL: its structure and function
International Congress Series 1233 (2002) 251 – 257
Heme-based oxygen sensor protein FixL: its structure and function Yoshitsugu Shiro*, Hiro Nakamura RIKEN Harima Institute/SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
Abstract The crystal structure of the heme-binding domain of the bacterial O2 sensor protein FixL from ˚ resolution. Combining the structural data Rhizobium meliloti (RmFixL) was determined at 1.4-A with some spectroscopic, biochemical, and molecular biological data, we discussed the O2 sensing mechanism by FixL. We proposed that the interaction of the iron-bound O2 with its surroundings would be a key step in triggering the intramolecular signal transduction from the O2-binding heme domain to the catalytic histidine kinase domain. D 2002 Elsevier Science B.V. All rights reserved. Keywords: O2 sensor; Rhizobial bacteria; Hemoprotein; Histidine kinase
1. O2 sensor protein FixL of rhizobial bacteria The rhizobial FixL/FixJ system is a paradigm of biological O2 sensors, which senses low O2 tensions to regulate the expression of the genes involved in nitrogen fixation in the symbiotic anaerobic state within the plant root nodules [1]. FixL and FixJ have also been known to be a pair of the sensory histidine kinases and the cognate response regulator of the bacterial two-component regulatory systems [2]. FixL derived from Rhizobium meliloti (RmFixL) consists of a membrane-anchoring domain, a sensor domain, and a histidine kinase domain. The histidine kinase domain is further divided into an autophosphorylation subdomain containing a phospho-accepting His285 (H box) and a catalytic subdomain containing an ATP-binding site (N box and G box) [2]. Since the sensor domain contains a heme, the dissociation of O2 from the heme iron triggers autophosphorylation in the kinase domain at low O2 tension. At low O2 tensions, FixL autophosphorylates a conserved histidine residue in the H box and transfers the phosphoryl group to FixJ to express the *
Corresponding author. E-mail address: [email protected] (Y. Shiro).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 2 3 6 - 4
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fixK and nifA genes, whereas FixL represses the kinase activity under aerobic conditions (Fig. 1A). To fully understand the molecular mechanism of O2 sensing by FixL, we have to elucidate three major points. First is the structural change of the sensor domain upon O2 dissociation, which possibly triggers kinase activation. Second is how information on the O2 dissociation from the sensor domain is transferred to the kinase domain. Third is how the kinase domain is activated in response to the signal from the sensor domain.
Fig. 1. (A) Two-component signal transduction of O2 sensor proteins FixL and FixJ directs expression of the nitrogen fixation-related genes at low O2 tension. (B) Optical absorption spectra of FixL in the deoxy and the oxy forms.
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Histidine kinases in the two-component regulatory systems share the well-conserved kinase domains, and mechanism of intra- and intermolecular signaling following the initial ligand binding is likely to be common, although the sensor domains are individually different. Thus, FixL/FixJ is the most useful model system of the ubiquitous two-component regulatory systems because the O2-free form (kinase-active) and the O2-bound form (kinaseinactive) of FixL are spectroscopically distinguishable (Fig. 1B) [3]. In this study, we will describe the molecular architecture of the FixL/FixJ system based on our recent studies.
2. N-terminal membrane-anchoring domain of FixL Since the sensory histidine kinases sense the extracellular ligands, the sensor domains of many kinases are localized in the cytoplasmic membranes [2]. Like other sensory kinases, RmFixL contains the hydrophobic membrane-spanning segments at the Nterminus. However, the localization of FixL in the membranes seems nonessential because O2 is membrane-permeable and the O2-binding site (heme) is exposed to the cytoplasm. In fact, we revealed that a deletion of the membrane-spanning regions from RmFixL resulted in no significant defects on the signaling functions in vivo and in vitro. This observation was apparently consistent with the fact that Bradyrhizobium japonicum FixL (BjFixL) possesses the hydrophilic fragment at the N-terminus.
3. Oxygen-binding domain of FixL Since FixL contains a protoheme as an O2-binding site, its electronic absorption spectra in the ferrous O2-bound (oxy-) and O2-free (deoxy-) forms are quite similar to the corresponding ones of hemoglobin. Local structural changes of the heme and its vicinity caused by O2 association/dissociation have been supposed to initiate the intramolecular regulation of the kinase domain by analogy of the R-T state conversion in hemoglobin [4 –8]. Recently, we and the Ohio group independently reported crystal structures of the heme domains of FixL from R. meliloti and B. japonicum, respectively [9,10]. In Fig. 2, the monomer part of the dimerized RmFixL heme domain is illustrated. The dimer is formed through hydrophobic and electrostatic interactions of the N-terminal helices. Since the Nterminal helices are truncated, the crystal structure of the BjFixL heme domain was determined as a monomer. The core structure of the monomer was essentially the same between the two FixL heme domains. The core structure of the heme domain consists of four a-helices and five h-strands. The heme is embedded in a hydrophobic crevice composed of F-helix and the h-sheet of four strands. The imidazole of His194 from the F helix is coordinated to the heme iron as a fifth ligand. The sixth coordination site is vacant in the ferric resting state, but is very crowded due to some hydrophobic groups (Ile209, Leu230, Val232), as shown in Fig. 3. Then, we thought that the iron-bound O2 might directly interact with the amino acids. Therefore, we prepared some mutants of RmFixL, in which the hydrophobic residues stated above were replaced with others, and we measured their kinase activities. In addition, the resonance Raman spectra were observed to examine the FeO2 bond char-
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Fig. 2. Overall structures of the sensor domains of RmFixL.
Fig. 3. Heme vicinity of the RmFixL sensor domain.
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Fig. 4. The kinase activities of some mutants of FixL in the deoxy and the oxy forms.
acters in these mutants. As shown in Fig. 4, the kinase activities could not be regulated by O2 binding in the mutants. The resonance Raman experiments showed that the iron-bound O2 directly or indirectly interacts with its surroundings [9]. On the basis of these observations, we have proposed that the interaction between the iron-bound O2 and its surroundings might be related to the kinase activation of FixL [11]. On the other hand, Gonzalez and co-workers reported the heme-domain structure of BjFixL in the ferric high spin and low spin states. Their crystallographic data have revealed that flattering/doming of the heme plane by ligand binding causes changes of the hydrogen bonds and the salt bridges between the heme propionates and the ‘‘FG regulatory loop’’ in the distal pocket of the heme [10]. Therefore, they have proposed that the conformational change of the loop initially transduces the ligand-binding signal to the kinase domain. However, it has been recently revealed in the crystal structure of the O2-bound form of the BjFixL sensor domain that the hydrogen bond is newly formed between the iron-bound O2 and conserved arginine (Arg214) at the G helix [12]. This finding agreed with our idea stated above.
4. Autophosphorylation domain and catalytic domain of FixL The autophosphorylation subdomains (H box) and catalytic subdomains (N and G boxes) are much conserved among the sensory histidine kinases. While these domains of FixL have not been studied enough, functional and structural analyses are available from the osmosensor EnvZ protein. The H box is responsible for the dimerization of the kinases by forming a pair of the two helical bundles [13]. Furthermore, two conserved histidines are exposed outside in opposite directions to each other. This configuration enables the
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histidines to accept the phosphoryl group from each catalytic domain and transfers it to the response regulator. The catalytic domain contains the conserved aspargine and glycines to stabilize the bound ATP, and the overall fold is found in ATPases (DNA gyrase and Hsp90) to form a super family of ATP-driven enzymes [14]. ATP is hydrolysed at the catalytic domain of one monomer, and the phosphoryl group is transferred to the histidine of the other monomer (transphosphorylation). Recently, we found that ADP, which is a product of ATP hydrolysis, affects the O2 affinity of the heme domain of FixL (data no shown). This may be related to the intramolecular interaction between the heme and the kinase domains.
5. FixJ as a transcriptional regulator FixJ is phosphorylated at Asp54 of the N-terminal receiver domain. Interacting with individual receiver domains dimerizes phospho-FixJ. We recently determined the structure of the C-terminal half of FixJ by NMR and found that a helix-turn-helix motif like other transcriptional factors is contained for binding to the fixK and nifA promoters (data not shown).
6. Conclusion Only 15 years have passed since the involvement of protein phosphorylations in bacterial adaptive responses was first discovered, and one can recognize in the last decade of the 20th century that the two-component signal transduction systems are widely spread in prokaryotic and eukaryotic organisms. As mentioned above, little of the regulatory mechanisms have been revealed at molecular or atomic level. We believe that knowledge obtained from the FixL/FixJ system will shed novel and profound insights into the ubiquitous signal transducing machinery as well as O2 sensing in living cells.
Acknowledgements This work was supported by the RIKEN Structural Biology Program, the MR Science Research Program in RIKEN, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan.
References [1] H.M. Fischer, Genetic regulation of nitrogen fixation in rhizobia, Microbiol. Rev. 58 (1994) 352 – 386. [2] J.B. Stock, A.J. Ninfa, A.M. Stock, Protein phosphorylation and regulation of adaptive responses in bacteria, Microbiol. Rev. 53 (1989) 450 – 490. [3] M.A. Gilles-Gonzalez, G.S. Ditta, D.R. Helinski, A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti, Nature 350 (1991) 170 – 172.
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[4] M.A. Gilles-Gonzalez, G. Gonzalez, Regulation of the kinase activity of heme protein FixL from the twocomponent system FixL/FixJ of Rhizobium meliloti, J. Biol. Chem. 268 (1993) 16293 – 16297. [5] M.A. Gilles-Gonzalez, G. Gonzalez, M.F. Perutz, L. Kiger, M.C. Marden, C. Poyart, Heme-based sensors, exemplified by the kinase FixL, are a new class of heme protein with distinctive ligand binding and autoxidation, Biochemistry 33 (1994) 8067 – 8073. [6] M.A. Gilles-Gonzalez, G. Gonzalez, M.F. Perutz, Kinase activity of oxygen sensor FixL depends on the spin state of its heme iron, Biochemistry 34 (1995) 232 – 236. [7] K. Tamura, H. Nakamura, Y. Tanaka, S. Oue, K. Tsukamoto, M. Nomura, T. Tsuchiya, S. Adachi, S. Takahashi, T. Iizuka, Y. Shiro, Nature of endogenous ligand binding to heme iron in oxygen sensor FixL, J. Am. Chem. Soc. 118 (1996) 9434 – 9435. [8] H. Miyatake, M. Mukai, S. Adachi, H. Nakamura, K. Tamura, T. Iizuka, Y. Shiro, R.W. Strange, S.S. Hasnain, 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. 274 (1999) 23176 – 23184. [9] H. Miyatake, M. Mukai, S.Y. Park, S. Adachi, K. Tamura, H. Nakamura, K. Nakamura, T. Tsuchiya, T. Iizuka, Y. Shiro, Sensory mechanism of oxygen sensor FixL from Rhizobium meliloti: crystallographic, mutagenesis and resonance Raman spectroscopic studies, J. Mol. Biol. 301 (2000) 415 – 431. [10] W. Gong, B. Hao, S.S. Mansy, G. Gonzalez, M.A. Gilles-Gonzalez, M.K. Chan, Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 15177 – 15182. [11] M. Mukai, K. Nakamura, H. Nakamura, T. Iizuka, Y. Shiro, Roles of Ile209 and Ile210 on the heme pocket structure and regulation of histidine kinase activity of oxygen sensor FixL from Rhizobium meliloti, Biochemistry 39 (2000) 13810 – 13816. [12] W. Gong, B. Hao, M.K. Chan, New mechanistic insights from structural studies of the oxygen-sensing domain of Bradyrhizobium japonicum FixL, Biochemistry 39 (2000) 3955 – 3962. [13] C. Tomomori, T. Tanaka, R. Dutta, H. Park, S.K. Saha, Y. Zhu, R. Ishima, D. Liu, K.I. Tong, H. Kurokawa, H. Qian, M. Inouye, M. Ikura, Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ, Nat. Struct. Biol. 6 (1999) 729 – 734. [14] T. Tanaka, S.K. Saha, C. Tomomori, R. Ishima, D. Liu, K.I. Tong, H. Park, R. Dutta, L. Qin, M.B. Swindells, T. Yamazaki, A.M. Ono, M. Kainosho, M. Inouye, M. Ikura, NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ, Nature 396 (1998) 88 – 92.
Heme-based oxygen sensor protein FixL: its structure and function Yoshitsugu Shiro*, Hiro Nakamura RIKEN Harima Institute/SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan
Abstract The crystal structure of the heme-binding domain of the bacterial O2 sensor protein FixL from ˚ resolution. Combining the structural data Rhizobium meliloti (RmFixL) was determined at 1.4-A with some spectroscopic, biochemical, and molecular biological data, we discussed the O2 sensing mechanism by FixL. We proposed that the interaction of the iron-bound O2 with its surroundings would be a key step in triggering the intramolecular signal transduction from the O2-binding heme domain to the catalytic histidine kinase domain. D 2002 Elsevier Science B.V. All rights reserved. Keywords: O2 sensor; Rhizobial bacteria; Hemoprotein; Histidine kinase
1. O2 sensor protein FixL of rhizobial bacteria The rhizobial FixL/FixJ system is a paradigm of biological O2 sensors, which senses low O2 tensions to regulate the expression of the genes involved in nitrogen fixation in the symbiotic anaerobic state within the plant root nodules [1]. FixL and FixJ have also been known to be a pair of the sensory histidine kinases and the cognate response regulator of the bacterial two-component regulatory systems [2]. FixL derived from Rhizobium meliloti (RmFixL) consists of a membrane-anchoring domain, a sensor domain, and a histidine kinase domain. The histidine kinase domain is further divided into an autophosphorylation subdomain containing a phospho-accepting His285 (H box) and a catalytic subdomain containing an ATP-binding site (N box and G box) [2]. Since the sensor domain contains a heme, the dissociation of O2 from the heme iron triggers autophosphorylation in the kinase domain at low O2 tension. At low O2 tensions, FixL autophosphorylates a conserved histidine residue in the H box and transfers the phosphoryl group to FixJ to express the *
Corresponding author. E-mail address: [email protected] (Y. Shiro).
0531-5131/02 D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 5 3 1 - 5 1 3 1 ( 0 2 ) 0 0 2 3 6 - 4
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fixK and nifA genes, whereas FixL represses the kinase activity under aerobic conditions (Fig. 1A). To fully understand the molecular mechanism of O2 sensing by FixL, we have to elucidate three major points. First is the structural change of the sensor domain upon O2 dissociation, which possibly triggers kinase activation. Second is how information on the O2 dissociation from the sensor domain is transferred to the kinase domain. Third is how the kinase domain is activated in response to the signal from the sensor domain.
Fig. 1. (A) Two-component signal transduction of O2 sensor proteins FixL and FixJ directs expression of the nitrogen fixation-related genes at low O2 tension. (B) Optical absorption spectra of FixL in the deoxy and the oxy forms.
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Histidine kinases in the two-component regulatory systems share the well-conserved kinase domains, and mechanism of intra- and intermolecular signaling following the initial ligand binding is likely to be common, although the sensor domains are individually different. Thus, FixL/FixJ is the most useful model system of the ubiquitous two-component regulatory systems because the O2-free form (kinase-active) and the O2-bound form (kinaseinactive) of FixL are spectroscopically distinguishable (Fig. 1B) [3]. In this study, we will describe the molecular architecture of the FixL/FixJ system based on our recent studies.
2. N-terminal membrane-anchoring domain of FixL Since the sensory histidine kinases sense the extracellular ligands, the sensor domains of many kinases are localized in the cytoplasmic membranes [2]. Like other sensory kinases, RmFixL contains the hydrophobic membrane-spanning segments at the Nterminus. However, the localization of FixL in the membranes seems nonessential because O2 is membrane-permeable and the O2-binding site (heme) is exposed to the cytoplasm. In fact, we revealed that a deletion of the membrane-spanning regions from RmFixL resulted in no significant defects on the signaling functions in vivo and in vitro. This observation was apparently consistent with the fact that Bradyrhizobium japonicum FixL (BjFixL) possesses the hydrophilic fragment at the N-terminus.
3. Oxygen-binding domain of FixL Since FixL contains a protoheme as an O2-binding site, its electronic absorption spectra in the ferrous O2-bound (oxy-) and O2-free (deoxy-) forms are quite similar to the corresponding ones of hemoglobin. Local structural changes of the heme and its vicinity caused by O2 association/dissociation have been supposed to initiate the intramolecular regulation of the kinase domain by analogy of the R-T state conversion in hemoglobin [4 –8]. Recently, we and the Ohio group independently reported crystal structures of the heme domains of FixL from R. meliloti and B. japonicum, respectively [9,10]. In Fig. 2, the monomer part of the dimerized RmFixL heme domain is illustrated. The dimer is formed through hydrophobic and electrostatic interactions of the N-terminal helices. Since the Nterminal helices are truncated, the crystal structure of the BjFixL heme domain was determined as a monomer. The core structure of the monomer was essentially the same between the two FixL heme domains. The core structure of the heme domain consists of four a-helices and five h-strands. The heme is embedded in a hydrophobic crevice composed of F-helix and the h-sheet of four strands. The imidazole of His194 from the F helix is coordinated to the heme iron as a fifth ligand. The sixth coordination site is vacant in the ferric resting state, but is very crowded due to some hydrophobic groups (Ile209, Leu230, Val232), as shown in Fig. 3. Then, we thought that the iron-bound O2 might directly interact with the amino acids. Therefore, we prepared some mutants of RmFixL, in which the hydrophobic residues stated above were replaced with others, and we measured their kinase activities. In addition, the resonance Raman spectra were observed to examine the FeO2 bond char-
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Fig. 2. Overall structures of the sensor domains of RmFixL.
Fig. 3. Heme vicinity of the RmFixL sensor domain.
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Fig. 4. The kinase activities of some mutants of FixL in the deoxy and the oxy forms.
acters in these mutants. As shown in Fig. 4, the kinase activities could not be regulated by O2 binding in the mutants. The resonance Raman experiments showed that the iron-bound O2 directly or indirectly interacts with its surroundings [9]. On the basis of these observations, we have proposed that the interaction between the iron-bound O2 and its surroundings might be related to the kinase activation of FixL [11]. On the other hand, Gonzalez and co-workers reported the heme-domain structure of BjFixL in the ferric high spin and low spin states. Their crystallographic data have revealed that flattering/doming of the heme plane by ligand binding causes changes of the hydrogen bonds and the salt bridges between the heme propionates and the ‘‘FG regulatory loop’’ in the distal pocket of the heme [10]. Therefore, they have proposed that the conformational change of the loop initially transduces the ligand-binding signal to the kinase domain. However, it has been recently revealed in the crystal structure of the O2-bound form of the BjFixL sensor domain that the hydrogen bond is newly formed between the iron-bound O2 and conserved arginine (Arg214) at the G helix [12]. This finding agreed with our idea stated above.
4. Autophosphorylation domain and catalytic domain of FixL The autophosphorylation subdomains (H box) and catalytic subdomains (N and G boxes) are much conserved among the sensory histidine kinases. While these domains of FixL have not been studied enough, functional and structural analyses are available from the osmosensor EnvZ protein. The H box is responsible for the dimerization of the kinases by forming a pair of the two helical bundles [13]. Furthermore, two conserved histidines are exposed outside in opposite directions to each other. This configuration enables the
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histidines to accept the phosphoryl group from each catalytic domain and transfers it to the response regulator. The catalytic domain contains the conserved aspargine and glycines to stabilize the bound ATP, and the overall fold is found in ATPases (DNA gyrase and Hsp90) to form a super family of ATP-driven enzymes [14]. ATP is hydrolysed at the catalytic domain of one monomer, and the phosphoryl group is transferred to the histidine of the other monomer (transphosphorylation). Recently, we found that ADP, which is a product of ATP hydrolysis, affects the O2 affinity of the heme domain of FixL (data no shown). This may be related to the intramolecular interaction between the heme and the kinase domains.
5. FixJ as a transcriptional regulator FixJ is phosphorylated at Asp54 of the N-terminal receiver domain. Interacting with individual receiver domains dimerizes phospho-FixJ. We recently determined the structure of the C-terminal half of FixJ by NMR and found that a helix-turn-helix motif like other transcriptional factors is contained for binding to the fixK and nifA promoters (data not shown).
6. Conclusion Only 15 years have passed since the involvement of protein phosphorylations in bacterial adaptive responses was first discovered, and one can recognize in the last decade of the 20th century that the two-component signal transduction systems are widely spread in prokaryotic and eukaryotic organisms. As mentioned above, little of the regulatory mechanisms have been revealed at molecular or atomic level. We believe that knowledge obtained from the FixL/FixJ system will shed novel and profound insights into the ubiquitous signal transducing machinery as well as O2 sensing in living cells.
Acknowledgements This work was supported by the RIKEN Structural Biology Program, the MR Science Research Program in RIKEN, and by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Culture, and Sports of Japan.
References [1] H.M. Fischer, Genetic regulation of nitrogen fixation in rhizobia, Microbiol. Rev. 58 (1994) 352 – 386. [2] J.B. Stock, A.J. Ninfa, A.M. Stock, Protein phosphorylation and regulation of adaptive responses in bacteria, Microbiol. Rev. 53 (1989) 450 – 490. [3] M.A. Gilles-Gonzalez, G.S. Ditta, D.R. Helinski, A haemoprotein with kinase activity encoded by the oxygen sensor of Rhizobium meliloti, Nature 350 (1991) 170 – 172.
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[4] M.A. Gilles-Gonzalez, G. Gonzalez, Regulation of the kinase activity of heme protein FixL from the twocomponent system FixL/FixJ of Rhizobium meliloti, J. Biol. Chem. 268 (1993) 16293 – 16297. [5] M.A. Gilles-Gonzalez, G. Gonzalez, M.F. Perutz, L. Kiger, M.C. Marden, C. Poyart, Heme-based sensors, exemplified by the kinase FixL, are a new class of heme protein with distinctive ligand binding and autoxidation, Biochemistry 33 (1994) 8067 – 8073. [6] M.A. Gilles-Gonzalez, G. Gonzalez, M.F. Perutz, Kinase activity of oxygen sensor FixL depends on the spin state of its heme iron, Biochemistry 34 (1995) 232 – 236. [7] K. Tamura, H. Nakamura, Y. Tanaka, S. Oue, K. Tsukamoto, M. Nomura, T. Tsuchiya, S. Adachi, S. Takahashi, T. Iizuka, Y. Shiro, Nature of endogenous ligand binding to heme iron in oxygen sensor FixL, J. Am. Chem. Soc. 118 (1996) 9434 – 9435. [8] H. Miyatake, M. Mukai, S. Adachi, H. Nakamura, K. Tamura, T. Iizuka, Y. Shiro, R.W. Strange, S.S. Hasnain, 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. 274 (1999) 23176 – 23184. [9] H. Miyatake, M. Mukai, S.Y. Park, S. Adachi, K. Tamura, H. Nakamura, K. Nakamura, T. Tsuchiya, T. Iizuka, Y. Shiro, Sensory mechanism of oxygen sensor FixL from Rhizobium meliloti: crystallographic, mutagenesis and resonance Raman spectroscopic studies, J. Mol. Biol. 301 (2000) 415 – 431. [10] W. Gong, B. Hao, S.S. Mansy, G. Gonzalez, M.A. Gilles-Gonzalez, M.K. Chan, Structure of a biological oxygen sensor: a new mechanism for heme-driven signal transduction, Proc. Natl. Acad. Sci. U. S. A. 95 (1998) 15177 – 15182. [11] M. Mukai, K. Nakamura, H. Nakamura, T. Iizuka, Y. Shiro, Roles of Ile209 and Ile210 on the heme pocket structure and regulation of histidine kinase activity of oxygen sensor FixL from Rhizobium meliloti, Biochemistry 39 (2000) 13810 – 13816. [12] W. Gong, B. Hao, M.K. Chan, New mechanistic insights from structural studies of the oxygen-sensing domain of Bradyrhizobium japonicum FixL, Biochemistry 39 (2000) 3955 – 3962. [13] C. Tomomori, T. Tanaka, R. Dutta, H. Park, S.K. Saha, Y. Zhu, R. Ishima, D. Liu, K.I. Tong, H. Kurokawa, H. Qian, M. Inouye, M. Ikura, Solution structure of the homodimeric core domain of Escherichia coli histidine kinase EnvZ, Nat. Struct. Biol. 6 (1999) 729 – 734. [14] T. Tanaka, S.K. Saha, C. Tomomori, R. Ishima, D. Liu, K.I. Tong, H. Park, R. Dutta, L. Qin, M.B. Swindells, T. Yamazaki, A.M. Ono, M. Kainosho, M. Inouye, M. Ikura, NMR structure of the histidine kinase domain of the E. coli osmosensor EnvZ, Nature 396 (1998) 88 – 92.