CO-dependent Activity-controlling Mechanism of Heme-containing CO-sensor Protein, Neuronal PAS Domain Protein 2

Abstract

Neuronal PAS domain protein 2, which was recently established to be a heme protein, acts as a CO-dependent transcription factor. The protein consists of the basic helix-loop-helix domain and two heme-containing PAS domains (PAS-A and PAS-B). In this study, we prepared wild type and mutants of the isolated PAS-A domain and measured resonance Raman spectra of these proteins. Upon excitation of the Raman spectrum at 363.8 nm, a band assignable to Fe 3؉ -S stretching was observed at 334 cm ؊1 for the ferric wild type protein; in contrast, this band was drastically weaker in the spectrum of C170A, suggesting that Cys 170 is an axial ligand of the ferric heme. The Raman spectrum of the reduced form of wild type was mainly of six-coordinate low spin, and the 11 band, which is sensitive to the donor strength of the axial ligand, was lower than that of reduced cytochrome c 3 , suggesting coordination of a strong ligand and thus a deprotonated His. In the reduced forms of H119A and H171A, the five-coordinate species became more prevalent, whereas no such changes were observed for C170A, indicating that His In recent years, a variety of heme-containing gas sensor proteins have been discovered in different species, from bacteria to mammals (1-3). In these proteins, a change in the concentration of gas molecules such as NO, O 2 , or CO is detected by a heme group and transduced to the functional domain as a signal, leading to modulation of protein activity. The hemebased gas sensor proteins discovered so far are listed in Table I. NO is a signaling molecule involved in vasodilation and neuronal transmission (4, 5). Soluble guanylate cyclase (sGC) 1 is a well known heme-based NO sensor protein. Upon binding of NO to sGC, the iron-histidine bond in the N-terminal region, the sole covalent linkage between the heme and protein, is cleaved. The bond cleavage induces conformational changes, resulting in a 400-fold increase in GC activity in the C-terminal region (6). Heme-regulated eukaryotic initiation factor 2␣ kinase (HRI) also forms a five-coordinated NO-heme complex via Fe-His bond cleavage, resulting in activation by NO (7). The O 2 -sensing proteins identified so far include FixL (8), DOS (9), PDEA1 (2), and HemAT (10, 11). The sensory domains of FixL and Ec DOS belong to the PAS 2 superfamily. FixL is a hemebased oxygen sensor involved in the regulation of expression of nitrogen fixation genes in response to O 2 concentration (8, 12, 13). Under low O 2 concentrations, FixL is autophosphorylated at a histidine residue and transfers it to FixJ, whereas a high concentration of O 2 suppresses kinase activity (8). Ec DOS is also an O 2 (and/or redox) sensor protein identified in Escherichia coli that exhibits phosphodiesterase activity in an O 2 -dependent (and/or redox-dependent) manner (9, 14). CooA was the first CO sensor protein identified from a purple nonsulfur photosynthetic bacterium, Rhodospirillum rubrum (15, 16). When CO binds to heme, the accompanying conformational changes induce binding of CooA to its target DNA ...

CO metabolism, sensing, and signaling

“…The best known CO-sensors is CooA, a homodimeric heme-containing protein, which regulates the CO oxidation system in the photosynthetic bacterium Rodospirillum rubrum [59][60][61][62], and the mammalian neuronal (N) period circadian protein (Per)-aryl hydrocarbon receptor nuclear translocator protein (Arnt) -single-minded protein (Sim) protein 2 (NPAS2), a member of the bHLH family of transcription factors expressed in the forebrain, which is a CO-dependent regulator of the circadian rhythm [59,60,63].…”

“…The mammalian NPAS2, together with its binding partner brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1), a bHLH transcription factor, binds DNA as an obligatory heterodimer [63,67]. The transcriptional activity of the NPAS2-BMAL1 heterodimer is modulated by CO and by the cellular redox balance (i.e., the NADPH/NADP molar ratio) [59,63].…”

“…The transcriptional activity of the NPAS2-BMAL1 heterodimer is modulated by CO and by the cellular redox balance (i.e., the NADPH/NADP molar ratio) [59,63]. The NPAS2 monomer contains two hemes located in the PAS domains (named PAS-A and PAS-B) and representing the CO-binding sites; both hemes are six-coordinated in the ''resting state'' [60,63].…”

“…The transcriptional activity of the NPAS2-BMAL1 heterodimer is modulated by CO and by the cellular redox balance (i.e., the NADPH/NADP molar ratio) [59,63]. The NPAS2 monomer contains two hemes located in the PAS domains (named PAS-A and PAS-B) and representing the CO-binding sites; both hemes are six-coordinated in the ''resting state'' [60,63]. Remarkably, CO binding to the heme-PAS domains, leading to the ''inactive state,'' inhibits the DNA binding activity of holo-PAS2 and leads to the formation of inactive BMAL1 homodimers instead of active NPAS2-BMAL1 heterodimers [63].…”

“…The NPAS2 monomer contains two hemes located in the PAS domains (named PAS-A and PAS-B) and representing the CO-binding sites; both hemes are six-coordinated in the ''resting state'' [60,63]. Remarkably, CO binding to the heme-PAS domains, leading to the ''inactive state,'' inhibits the DNA binding activity of holo-PAS2 and leads to the formation of inactive BMAL1 homodimers instead of active NPAS2-BMAL1 heterodimers [63]. In vivo, this results in the negative modulation of the day-night cycle [67].…”