title: Heme-binding characteristics of the isolated PAS-A domain of mouse Per2, a transcriptional regulatory factor associated with circadian rhythms.
abstract: 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.
authors.affiliation: Institute of Multidisciplinary Research for Advanced Materials, Tohoku UniVersity, Katahira, Sendai 980-8577, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Kitanishi
authors.forename: Jotaro
authors.initials: J
authors.lastname: Igarashi
authors.forename: Koya
authors.initials: K
authors.lastname: Hayasaka
authors.forename: Naoki
authors.initials: N
authors.lastname: Hikage
authors.forename: Islam
authors.initials: I
authors.lastname: Saiful
authors.forename: Seigo
authors.initials: S
authors.lastname: Yamauchi
authors.forename: Takeshi
authors.initials: T
authors.lastname: Uchida
authors.forename: Koichiro
authors.initials: K
authors.lastname: Ishimori
authors.forename: Toru
authors.initials: T
authors.lastname: Shimizu
mesh_terms: D000818:Animals; D051792:Basic Helix-Loop-Helix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D001921:Brain / Q000502:physiology; D018797:Cell Cycle Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002940:Circadian Rhythm / Q000502:physiology*; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism; D017931:DNA Primers; D006418:Heme / Q000737:chemistry / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008628:Mercury / Q000378:metabolism; D051379:Mice; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism*; D056950:Period Circadian Proteins; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013059:Spectrum Analysis, Raman; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D018797:Cell Cycle Proteins; D003545:Cysteine; D017931:DNA Primers; D006418:Heme; D007501:Iron; D008628:Mercury; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D010446:Peptide Fragments; D056950:Period Circadian Proteins; D011994:Recombinant Proteins; D014157:Transcription Factors; C491259:Npas2 protein, mouse; C513312:Per2 protein, mouse