Structural Characterization of the Proximal and Distal Histidine Environment of Cytoglobin and Neuroglobin Hitomi Sawai, ‡,§ Masatomo Makino, ‡,§ Yasuhisa Mizutani, | Takehiro Ohta, ⊥ Hiroshi Sugimoto, § Tadayuki Uno, @ Norifumi Kawada, # Katsutoshi Yoshizato, + Teizo Kitagawa, ⊥ and Yoshitsugu Shiro* ,§ Department of Life Science, Graduate School of Science, Himeji Institute of Technology/UniVersity of Hyogo, 3-2-1 Kouto, Kamigori-cho, Ako, Hyogo 678-1297, Japan, RIKEN Harima Institute/SPring-8, 1-1-1 Kouto, Mikazuki-cho, Sayo, Hyogo 679-5148, Japan, Molecular Photoscience Research Center, Kobe UniVersity, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan, Center for IntegratiVe Bioscience, Okazaki National Research Institutes, Myodaiji, Okazaki, Aichi 444-8787, Japan, Graduate School of Pharmaceutical Sciences, Osaka UniVersity, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan, Department of Hepatology, Graduate School of Medicine, Osaka City UniVersity, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan, DeVelopmental Biology Laboratory and 21st Century COE Program for AdVanced Radiation Casualty Medicine, Department of Biological Science, Graduate School of Science, Hiroshima UniVersity, 1-3-1 Kagamiyama, Higashihiroshima, Hiroshima 739-8526, Japan, and Yoshizato Project, CooperatiVe Link of Unique Science and Technology for Economy ReVitalization (Japan Science and Technology Organization), Hiroshima Prefectural Institute of Industrial Science and Technology, 3-10-32, Kagamiyama, Higashihiroshima, Hiroshima 739-006, Japan ReceiVed May 27, 2005; ReVised Manuscript ReceiVed August 1, 2005 ABSTRACT: Cytoglobin (Cgb) and neuroglobin (Ngb) are the first examples of hexacoordinated globins from humans and other vertebrates in which a histidine (His) residue at the sixth position of the heme iron is an endogenous ligand in both the ferric and ferrous forms. Static and time-resolved resonance Raman and FT-IR spectroscopic techniques were applied in examining the structures in the heme environment of these globins. Picosecond time-resolved resonance Raman (ps-TR 3 ) spectroscopy of transient five-coordinate heme species produced by the photolysis of carbon monoxide (CO) adducts of Cgb and Ngb showed Fe-His stretching (ν Fe-His ) bands at 229 and 221 cm -1 , respectively. No time-dependent shift in the ν Fe-His band of Cgb and Ngb was detected in the 20-1000 ps time domain, in contrast to the case of myoglobin (Mb). These spectroscopic data, combined with previously reported crystallographic data, suggest that the structure of the heme pocket in Cgb and Ngb is altered upon CO binding in a manner different from that of Mb and that the scales of the structural alteration are different for Cgb and Ngb. The structural property of the heme distal side of the ligand-bound forms was investigated by observing the sets of (ν Fe-CO , ν C-O , δ Fe-C-O ) and (ν Fe-NO , ν N-O , δ Fe-N-O ) for the CO and nitric oxide (NO) complexes of Cgb and Ngb. A comparison of the spectra of some distal mutants of Cgb (H81A, H81V, R84A, R84K, and R84T) and Ngb (H64A, H64V, K67A, K67R, and K67T) showed that the CO adducts of Cgb and Ngb contained three conformers and that the distal His (His81 in Cgb and His64 in Ngb) mainly contributes to the interconversion of the conformers. These structural characteristics of Cgb and Ngb are discussed in relation to their ligand binding and physiological properties. Heme-containing globin proteins are widely distributed in biological systems, including bacteria, fungi, protists, plants, and animals (1). In vertebrates, tetrameric hemoglobin (Hb) 1 present in erythrocytes plays a role in supplying oxygen (O 2 ) to the body (2), while monomeric and muscle-specific myoglobin (Mb) serves as a sourse of intracellular O 2 storage to possibly enhance the diffusion of O 2 to the mitochondria and/or is presumably involved in the detoxification of NO (3). In addition to Hb and Mb, two novel members, neuroglobin (Ngb) (4) and cytoglobin (Cgb) (5-7), have recently been added to the vertebrate globin family. Cgb is also known as the stellate cell activation-associated protein (STAP) (6, 7) or histoglobin (9). This protein is present in virtually all tissues. On the other hand, Ngb is present in cells of the vertebrate central and peripheral nervous system, the retina (8), and also endocrine tissue (4). The primary sequences of Cgb and Ngb are only distantly related to Hb and Mb, the level of identity being ∼25%, but some amino acid residues are highly conserved in the vertebrate globin family (10). Since the conserved residues of Mb and Hb are located around the heme active sites and are important in the binding of external ligands to the heme iron, it has been suggested that Ngb and Cgb are possibly involved in the binding of O 2 in the distributed cells. Although the physi- * To whom correspondence should be addressed. Telephone: +81- 791-58-2817. Fax: +81-791-58-2818. E-mail: yshiro@riken.jp. ‡ Himeji Institute of Technology/University of Hyogo. § RIKEN Harima Institute/SPring-8. | Kobe University. ⊥ Okazaki National Research Institutes. @ Osaka University. # Osaka City University. + Hiroshima University and Hiroshima Prefectural Institute of Industrial Science and Technology. 1 Abbreviations: Cgb, cytoglobin; Ngb, neuroglobin; Mb, myoglobin; Hb, hemoglobin; TR 3 , time-resolved resonance Raman; FT-IR, Fourier transform infrared; CO, carbon monoxide; NO, nitric oxide; νFe-His, Fe-His stretching mode. 13257 Biochemistry 2005, 44, 13257-13265 10.1021/bi050997o CCC: $30.25 © 2005 American Chemical Society Published on Web 09/13/2005