期刊
JOURNAL OF BIOCHEMISTRY
卷 140, 期 4, 页码 561-571出版社
OXFORD UNIV PRESS
DOI: 10.1093/jb/mvj189
关键词
cytochrome b(5); diethylpyrocarbonate; histidyl residue; MALDI-TOF; N-carbethoxylation
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-epsilon 2 coordination bond, and protonation of the N delta(1) position by forming a hydrogen bond with its immediate surroundings. However, once N-carbethoxylation at the N-epsilon 2 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-epsilon 1 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-delta 1 atom works as a hydrogen acceptor in a strong hydrogen-bond and the other N-epsilon 2 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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