Characterization of heme-coordinating histidyl residues of cytochrome b5 based on the reactivity with diethylpyrocarbonate: a mechanism for the opening of axial imidazole rings.
We investigated the reactivity of heme-coordinating imidazole with diethylpyrocarbonate using a soluble domain of cytochrome b(5). Analyses with various spectroscopic methods including MALDI-TOF-MS indicated that two axial His residues (His44 and His68) of cytochrome b(5) were protected from the modification by several factors, i.e., limited steric exposure of the axial imidazole to the solvent, the Fe-N(epsilon2) coordination bond, and protonation of the N(delta1) position by forming a hydrogen bond with its immediate surroundings. However, once N-carbethoxylation at the N(epsilon2) 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 N(epsilon1) 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 N(delta1) atom works as a hydrogen acceptor in a strong hydrogen-bond and the other N(epsilon2) 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.
Nakanishi
Nobuyuki
N
Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501.
Takeuchi
Fusako
F
Okamoto
Hidetsugu
H
Tamura
Atsuo
A
Hori
Hiroshi
H
Tsubaki
Motonari
M
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D015786:Cytochromes b5 / Q000737:chemistry*; D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000737:chemistry*; D006639:Histidine / Q000737:chemistry*; D006801:Humans; D006860:Hydrogen Bonding; D007093:Imidazoles / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D007093:Imidazoles; D006418:Heme; D006639:Histidine; D015786:Cytochromes b5; D004047:Diethyl Pyrocarbonate