Jotaro Igarashi

Elucidation of the heme binding site of heme-regulated eukaryotic initiation factor 2alpha kinase and the role of the regulatory motif in heme sensing by spectroscopic and catalytic studies of mutant proteins.

Heme-regulated eukaryotic initiation factor 2α (eIF2α) kinase (HRI) functions in response to the heme iron concentration. At the appropriate heme iron concentrations under normal conditions, HRI function is suppressed by binding of the heme iron. Conversely, upon heme iron shortage, HRI autophosphorylates and subsequently phosphorylates the substrate, eIF2α, leading to the termination of protein synthesis. The molecular mechanism of heme sensing by HRI, including identification of the specific binding site, remains to be established. In the present study we demonstrate that His-119/His-120 and Cys-409 are the axial ligands for the Fe(III)-protoporphyrin IX complex (hemin) in HRI, based on spectral data on site-directed mutant proteins. Cys-409 is part of the heme-regulatory Cys-Pro motif in the kinase domain. A P410A full-length mutant protein displayed loss of heme iron affinity. Surprisingly, inhibitory effects of the heme iron on catalysis and changes in the heme dissociation rate constants in full-length His-119/His-120 and Cys-409 mutant proteins were marginally different to wild type. In contrast, heme-induced inhibition of Cys-409 mutants of the isolated kinase domain and N-terminal-truncated proteins was substantially weaker than that of the full-length enzyme. A pulldown assay disclosed heme-dependent interactions between the N-terminal and kinase domains. Accordingly, we propose that heme regulation is induced by interactions between heme and the catalytic domain in conjunction with global tertiary structural changes at the N-terminal domain that accompany heme coordination and not merely by coordination of the heme iron with amino acids on the protein surface.

Selective neuronal nitric oxide synthase inhibitors and the prevention of cerebral palsy

To design a new class of selective neuronal nitric oxide synthase (NOS) inhibitors, and demonstrate that administration in a rabbit model for cerebral palsy (CP) prevents hypoxia‐ischemia–induced deaths and reduces the number of newborn kits exhibiting signs of CP.

Important roles of Tyr43 at the putative heme distal side in the oxygen recognition and stability of the Fe(II)-O2 complex of YddV, a globin-coupled heme-based oxygen sensor diguanylate cyclase.

YddV from Escherichia coli (Ec) is a novel globin-coupled heme-based oxygen sensor protein displaying diguanylate cyclase activity in response to oxygen availability. In this study, we quantified the turnover numbers of the active [Fe(III), 0.066 min(-1); Fe(II)-O(2) and Fe(II)-CO, 0.022 min(-1)] [Fe(III), Fe(III)-protoporphyrin IX complex; Fe(II), Fe(II)-protoporphyrin IX complex] and inactive forms [Fe(II) and Fe(II)-NO, <0.01 min(-1)] of YddV for the first time. Our data indicate that the YddV reaction is the rate-determining step for two consecutive reactions coupled with phosphodiesterase Ec DOS activity on cyclic di-GMP (c-di-GMP) [turnover number of Ec DOS-Fe(II)-O(2), 61 min(-1)]. Thus, O(2) binding and the heme redox switch of YddV appear to be critical factors in the regulation of c-di-GMP homeostasis. The redox potential and autoxidation rate of heme of the isolated heme domain of YddV (YddV-heme) were determined to be -17 mV versus the standard hydrogen electrode and 0.0076 min(-1), respectively. The Fe(II) complexes of Y43A and Y43L mutant proteins (residues at the heme distal side of the isolated heme-bound globin domain of YddV) exhibited very low O(2) affinities, and thus, their Fe(II)-O(2) complexes were not detected on the spectra. The O(2) dissociation rate constant of the Y43W protein was >150 s(-1), which is significantly larger than that of the wild-type protein (22 s(-1)). The autoxidation rate constants of the Y43F and Y43W mutant proteins were 0.069 and 0.12 min(-1), respectively, which are also markedly higher than that of the wild-type protein. The resonance Raman frequencies representing ν(Fe-O(2)) (559 cm(-1)) of the Fe(II)-O(2) complex and ν(Fe-CO) (505 cm(-1)) of the Fe(II)-CO complex of Y43F differed from those (ν(Fe-O(2)), 565 cm(-1); ν(Fe-CO), 495 cm(-1)) of the wild-type protein, suggesting that Tyr43 forms hydrogen bonds with both O(2) and CO molecules. On the basis of the results, we suggest that Tyr43 located at the heme distal side is important for the O(2) recognition and stability of the Fe(II)-O(2) complex, because the hydroxyl group of the residue appears to interact electrostatically with the O(2) molecule bound to the Fe(II) complex in YddV. Our findings clearly support a role of Tyr in oxygen sensing, and thus modulation of overall conversion from GTP to pGpG via c-di-GMP catalyzed by YddV and Ec DOS, which may be applicable to other globin-coupled oxygen sensor enzymes.

Heme-Binding Characteristics of the Isolated PAS-A Domain of Mouse Per2, a Transcriptional Regulatory Factor Associated with Circadian Rhythms

Neuronal PAS protein 2 (NPAS2), a heme-binding transcriptional regulatory factor, is involved in circadian rhythms. Period homologue (Per) is another important transcriptional regulatory factor that binds to cryptochrome (Cry). The resultant Per/Cry heterodimer interacts with the NPAS2/BMAL1 heterodimer to inhibit the transcription of Per and Cry. Previous cell biology experiments indicate that mouse Per2 (mPer2) is also a heme-binding protein, and heme shuttling between mPer2 and NPAS2 may regulate transcription. In the present study, we show that the isolated PAS-A domain of mPer2 (PAS-A-mPer2) binds the Fe(III) protoporphyrin IX complex (hemin) with a heme:protein stoichiometry of 1:1. Optical absorption and EPR spectroscopic findings suggest that the Fe(III)-bound PAS-A-mPer2 is a six-coordinated low-spin complex with Cys and an unknown axial ligand. A Hg (2+) binding study supports the theory that Cys is one of the axial ligands for Fe(III)-bound PAS-A-mPer2. The dissociation rate constant of the Fe(III) complex from PAS-A-mPer2 (6.3 x 10 (-4) s (-1)) was comparable to that of the heme-regulated inhibitor (HRI), a heme-sensor enzyme (1.5 x 10 (-3) s (-1)), but markedly higher than that of metmyoglobin (8.4 x 10 (-7) s (-1)). As confirmed by a Soret absorption spectral shift, heme transferred from the holo basic helix-loop-helix PAS-A of NPAS2 to apoPAS-A-mPer2. The Soret CD spectrum of the C215A mutant PAS-A-mPer2 protein was markedly different from that of the wild-type protein. On the basis of the data, we propose that PAS-A-mPer2 is a heme-sensor protein in which Cys215 is the heme axial ligand.

Identification and Functional and Spectral Characterization of a Globin-coupled Histidine Kinase from Anaeromyxobacter sp. Fw109-5

Background: Two-component signal transduction systems regulate important physiological functions in bacteria. Results: The Fe(III), Fe(II)-O2, and Fe(II)-CO complexes of a heme-bound globin-coupled histidine kinase from Anaeromyxobacter displayed autophosphorylation activity, whereas the Fe(II) complex was inactive. Conclusion: Gas binding and heme redox regulate the histidine kinase function. Significance: A novel heme-based globin-coupled oxygen sensor histidine kinase was identified and characterized. Two-component signal transduction systems regulate numerous important physiological functions in bacteria. In this study we have identified, cloned, overexpressed, and characterized a dimeric full-length heme-bound (heme:protein, 1:1 stoichiometry) globin-coupled histidine kinase (AfGcHK) from Anaeromyxobacter sp. strain Fw109-5 for the first time. The Fe(III), Fe(II)-O2, and Fe(II)-CO complexes of the protein displayed autophosphorylation activity, whereas the Fe(II) complex had no significant activity. A H99A mutant lost heme binding ability, suggesting that this residue is the heme proximal ligand. Moreover, His-183 was proposed as the autophosphorylation site based on the finding that the H183A mutant protein was not phosphorylated. The phosphate group of autophosphorylated AfGcHK was transferred to Asp-52 and Asp-169 of a response regulator, as confirmed from site-directed mutagenesis experiments. Based on the amino acid sequences and crystal structures of other globin-coupled oxygen sensor enzymes, Tyr-45 was assumed to be the O2 binding site at the heme distal side. The O2 dissociation rate constant, 0.10 s−1, was substantially increased up to 8.0 s−1 upon Y45L mutation. The resonance Raman frequencies representing νFe-O2 (559 cm−1) and νO-O (1149 cm−1) of the Fe(II)-O2 complex of Y45F mutant AfGcHK were distinct from those of the wild-type protein (νFe-O2, 557 cm−1; νO-O, 1141 cm−1), supporting the proposal that Tyr-45 is located at the distal side and forms hydrogen bonds with the oxygen molecule bound to the Fe(II) complex. Thus, we have successfully identified and characterized a novel heme-based globin-coupled oxygen sensor histidine kinase, AfGcHK, in this study.

Arg97 at the Heme-Distal Side of the Isolated Heme-Bound PAS Domain of a Heme-Based Oxygen Sensor from Escherichia coli (Ec DOS) Plays Critical Roles in Autoxidation and Binding to Gases, Particularly O2

The catalytic activity of heme-regulated phosphodiesterase from Escherichia coli (Ec DOS) on cyclic di-GMP is markedly enhanced upon binding of gas molecules, such as O2 and CO, to the heme iron complex in the sensor domain. Arg97 interacts directly with O2 bound to Fe(II) heme in the crystal structure of the isolated heme-bound sensor domain with the PAS structure (Ec DOS-PAS) and may thus be critical in ligand recognition. To establish the specific role of Arg97, we generated Arg97Ala, Arg97Glu, and Arg97Ile mutant Ec DOS-PAS proteins and examined binding to O2, CO, and cyanide, as well as redox potentials. The autoxidation rates of the Arg97Ala and Arg97Glu mutant proteins were up to 2000-fold higher, while the O2 dissociation rate constant for dissociation from the Fe(II)-O2 heme complex of the Arg97Ile mutant was 100-fold higher than that of the wild-type protein. In contrast, the redox potential values of the mutant proteins were only slightly different from that of the wild type (within 10 mV). Accordingly, we propose that Arg97 plays critical roles in recognition of the O2 molecule and redox switching by stabilizing the Fe(II)-O2 complex, thereby anchoring O2 to the heme iron and lowering the autoxidation rate to prevent formation of Fe(III) hemin species not regulated by gas molecules. Arg97 mutations significantly influenced interactions with the internal ligand Met95, during CO binding to the Fe(II) complex. Moreover, the binding behavior of cyanide to the Fe(III) complexes of the Arg mutant proteins was similar to that of O2, which is evident from the Kd values, suggestive of electrostatic interactions between cyanide and Arg97.

Spectroscopic and DNA-binding characterization of the isolated heme-bound basic helix–loop–helix-PAS-A domain of neuronal PAS protein 2 (NPAS2), a transcription activator protein associated with circadian rhythms

Neuronal PAS domain protein 2 (NPAS2) is a circadian rhythm‐associated transcription factor with two heme‐binding sites on two PAS domains. In the present study, we compared the optical absorption spectra, resonance Raman spectra, heme‐binding kinetics and DNA‐binding characteristics of the isolated fragment containing the N‐terminal basic helix–loop–helix (bHLH) of the first PAS (PAS‐A) domain of NPAS2 with those of the PAS‐A domain alone. We found that the heme‐bound bHLH‐PAS‐A domain mainly exists as a dimer in solution. The Soret absorption peak of the Fe(III) complex for bHLH‐PAS‐A (421 nm) was located at a wavelength 9 nm higher than for isolated PAS‐A (412 nm). The axial ligand trans to CO in bHLH‐PAS‐A appears to be His, based on the resonance Raman spectra. In addition, the rate constant for heme association with apo‐bHLH‐PAS (3.3 × 107 mol−1·s−1) was more than two orders of magnitude higher than for association with apo‐PAS‐A (< 105 mol−1·s−1). These results suggest that the bHLH domain assists in stable heme binding to NPAS2. Both optical and resonance Raman spectra indicated that the Fe(II)–NO heme complex is five‐coordinated. Using the quartz‐crystal microbalance method, we found that the bHLH‐PAS‐A domain binds specifically to the E‐box DNA sequence in the presence, but not in the absence, of heme. On the basis of these results, we discuss the mode of heme binding by bHLH‐PAS‐A and its potential role in regulating DNA binding.

Leu65 in the heme distal side is critical for the stability of the Fe(II)-O2 complex of YddV, a globin-coupled oxygen sensor diguanylate cyclase.

YddV is a globin-coupled oxygen sensor enzyme in that O(2) binding to the Fe(II) heme in the sensor domain substantially enhances its diguanylate cyclase activity. The Fe(III) heme-bound enzyme is also the active form. Amino acid sequence comparisons indicate that Leu65 is well conserved in globin-coupled oxygen sensor enzymes. Absorption spectra of the Fe(III) heme complexes of L65G, L65M, L65Q and L65T mutants of the isolated heme domain of YddV (YddV-heme) were substantially different from that of the wild-type protein. Specifically, Soret bands of the 6-coordinated high-spin Fe(III) complexes of mutant proteins (with H(2)O and His98 as axial ligands) were located at around 403-406 nm, distinct from that (391 nm) of the 5-coordinated high-spin Fe(III) complex of wild-type protein with His98 as the axial ligand. The autooxidation rate constant (>0.10 min(-1)) of the Fe(II)-O(2) complex of L65G was substantially higher than that (0.011 min(-1)) of the wild-type protein. Affinities of O(2) for the Fe(II) complexes of L65G and L65T were markedly higher than that for the wild-type protein. Thus, we suggest that the well-conserved Leu65 located in the heme distal side is critical for restricting water access to the heme distal side to avoid rapid autooxidation of YddV, which needs a stable Fe(II)-O(2) complex with a low autooxidation rate.

Spectroscopic characterization of the isolated heme-bound PAS-B domain of neuronal PAS domain protein 2 associated with circadian rhythms.

Neuronal PAS domain protein 2 (NPAS2) is an important transcription factor associated with circadian rhythms. This protein forms a heterodimer with BMAL1, which binds to the E‐box sequence to mediate circadian rhythm‐regulated transcription. NPAS2 has two PAS domains with heme‐binding sites in the N‐terminal portion. In this study, we overexpressed wild‐type and His mutants of the PAS‐B domain (residues 241–416) of mouse NPAS2 and then purified and characterized the isolated heme‐bound proteins. Optical absorption spectra of the wild‐type protein showed that the Fe(III), Fe(II) and Fe(II)–CO complexes are 6‐co‐ordinated low‐spin complexes. On the other hand, resonance Raman spectra indicated that both the Fe(III) and Fe(II) complexes contain mixtures of 5‐co‐ordinated high‐spin and 6‐co‐ordinated low‐spin complexes. Based on inverse correlation between νFe‐CO and νC‐O of the resonance Raman spectra, it appeared that the axial ligand trans to CO of the heme‐bound PAS‐B is His. Six His mutants (His266Ala, His289Ala, His300Ala, His302Ala, His329Ala, and His335Ala) were generated, and their optical absorption spectra were compared. The spectrum of the His335Ala mutant indicated that its Fe(III) complex is the 5‐co‐ordinated high‐spin complex, whereas, like the wild‐type, the complexes for the five other His mutants were 6‐co‐ordinated low‐spin complexes. Thus, our results suggest that one of the axial ligands of Fe(III) in PAS‐B is His335. Also, binding kinetics suggest that heme binding to the PAS‐B domain of NPAS2 is relatively weak compared with that of sperm whale myoglobin.

Heme-binding characteristics of the isolated PAS-B domain of mouse Per2, a transcriptional regulatory factor associated with circadian rhythms.

Mouse period homolog 2 (mPer2), an important transcriptional regulatory factor associated with circadian rhythms, is composed of two N-terminal PAS (PAS-A and PAS-B) domains and a C-terminal domain. The PAS-A domain of mPer2 binds the heme iron via a Cys axial ligand. A corresponding transcriptional regulatory factor, neuronal PAS 2 protein (NPAS2), also contains PAS-A and PAS-B domains at the N-terminus with heme-binding capability. In particular, the PAS-B domain appears important for protein-protein interactions critical for transcriptional regulation. In the present study, we examined the heme-binding characteristics of the isolated PAS-B domain of mPer2. Our experiments show that the Fe(III) heme binds the isolated PAS-B domain with a heme to protein stoichiometry of 1:1. The Fe(III) protein complex is suggested to consist of an admixture of 6-coordinated His-bound high-spin and low-spin complexes. Marked pH-dependent spectral changes were observed, in contrast to the spectrum of the Fe(III) bound PAS-A domain of mPer2, which appeared pH-resistant. Treatment with diethylpyrocarbonate abolished the heme-binding ability of this protein, supporting the proposal that His is the axial ligand. Heme dissociation was composed of two phases with rate constants of 4.3 × 10⁻⁴ s⁻¹ (50%) and 4.0 × 10⁻³ s⁻¹ (50%), which were markedly higher than that (1.5 × 10⁻⁷ s⁻¹) of the prototype heme protein, myoglobin. The Soret CD band of the H454A PAS-B mutant was significantly different from those of wild-type and other His mutant proteins, strongly suggesting that His454 is one of the axial ligands for the Fe(III) complex.