Journal of Biochemistry Advance Access published online on September 8, 2006
Journal of Biochemistry, doi:10.1093/jb/mvj189
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1 Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan
* To whom correspondence should be addressed. We investigated the reactivity of heme-coordinating imidazole with diethylpyrocarbonate using a soluble domain of cytochrome b5. Analyses with various spectroscopic methods including MALDI-TOF-MS indicated that two axial His residues (His44 and His68) of cytochrome b5 were protected from the modification by several factors, i.e., limited steric exposure of the axial imidazole to the solvent, the Fe-N
Received August 1, 2006
Accepted August 30, 2006
Regular Paper
Characterization of Heme-Coordinating Histidyl Residues of Cytochrome b5 Based on the Reactivity with Diethylpyrocarbonate: A Mechanism for the Opening of Axial Imidazole Rings
Nobuyuki Nakanishi 1, Fusako Takeuchi 1, Hidetsugu Okamoto 1, Atsuo Tamura 1, Hiroshi Hori 2, and Motonari Tsubaki 3 *
2 Division of Bioengineering, Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, Machikaneyama-cho, Toyonaka, Osaka 560-8531, Japan
3 Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan; CREST, JST, Japan
Motonari Tsubaki, E-mail: mtsubaki{at}kobe-u.ac.jp
Abstract 
2 coordination bond, and protonation of the N
1 position by forming a hydrogen bond with its immediate surroundings. However, once N-carbethoxylation at the N2 position of the axial His residues occurred with a higher concentration of diethylpyrocarbonate, displacement of heme prosthetic group from the protein moiety continued. Simultaneously, it facilitated the second N-carbethoxylation to take place at the N1 position of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. A similar mechanism seemed in operation for one non-axial His residue (His85), in which the N1 atom works as a hydrogen acceptor in a strong hydrogen-bond and the other N2 atom is in a protonated form, resulting in a formation of the ring-opened derivative upon treatment with a higher concentration of diethylpyrocarbonate. These results suggested that the use of diethylpyrocarbonate for MALDI-TOF-MS analysis might provide a unique method to characterize the protonation state of His residues and the strength of their hydrogen-bondings at the active site of enzymes.
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