CO-dependent activity-controlling mechanism of heme-containing CO-sensor protein, neuronal PAS domain protein

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 Fe3+-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 v(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(119) and His(171), but not Cys(170), are axial ligands in the ferrous heme. This means that ligand replacement from Cys to His occurs upon heme reduction. The v(Fe-CO) versus v(C-O) correlation indicates that a neutral His is a trans ligand of CO. Our results support a mechanism in which CO binding disrupts the hydrogen bonding of His(171) with surrounding amino acids, which induces conformational changes in the His(171)- Cys(170) moiety, leading to physiological signaling.