title: Spectroscopic characterization of the isolated heme-bound PAS-B domain of neuronal PAS domain protein 2 associated with circadian rhythms.
abstract: 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 nu(Fe-CO) and nu(C-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.
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Sendai, Japan.
authors.forename: Ryoji
authors.initials: R
authors.lastname: Koudo
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Kurokawa
authors.forename: Emiko
authors.initials: E
authors.lastname: Sato
authors.forename: Jotaro
authors.initials: J
authors.lastname: Igarashi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchida
authors.forename: Ikuko
authors.initials: I
authors.lastname: Sagami
authors.forename: Teizo
authors.initials: T
authors.lastname: Kitagawa
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins; D002850:Chromatography, Gel; D002940:Circadian Rhythm / Q000502:physiology*; D017931:DNA Primers; D006418:Heme / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013059:Spectrum Analysis, Raman / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D017931:DNA Primers; D006418:Heme; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C491259:Npas2 protein, mouse