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

作者：Hayasaka Koya; Kitanishi Kenichi; Igarashi Jotaro; Shimizu Toru^*

来源：Biochimica et Biophysica Acta-Proteins and Proteomics, 2011, 1814(2): 326-333.

DOI：10.1016/j.bbapap.2010.09.007

• Heme-binding protein
• PAS domain
• Circadian rhythm
• Transcriptional regulation

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 x 10(-4) s(-1) (50%) and 4.0 x 10(-3) s(-1) (50%), which were markedly higher than that (1.5 x 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.

• 出版日期2011-2