Journal of Biochemistry Advance Access originally published online on September 8, 2006
Journal of Biochemistry 2006 140(4):561-571; doi:10.1093/jb/mvj189
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© 2006 The Japanese Biochemical Society.
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Characterization of Heme-Coordinating Histidyl Residues of Cytochrome b5 Based on the Reactivity with Diethylpyrocarbonate: A Mechanism for the Opening of Axial Imidazole Rings
1 Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Kobe University, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501; 2 Division of Bioengineering, Department of Mechanical Science and Bioengineering, Graduate School of Engineering Science, Osaka University, Machikaneyama-cho, Toyonaka, Osaka 560-8531; and 3 CREST, JST
* To whom correspondence should be addressed. Tel/Fax: +81-78-803-6582, E-mail: mtsubaki{at}kobe-u.ac.jp
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
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.
1The majority of hemoproteins having His residues as an axial ligand are known to make the coordination to heme iron with their N2 atoms. Only a few exceptions to have an Fe-N1 bond are known, such as the c-type low-spin heme-I in the tetra-heme cytochorme c554 from the bacterium Nitrosomonas europaea (1) and the Met65His mutant of the heme domain of cellobiose dehydrogenase (2).
2Numbering of amino acid residues in the present study is different from the crystallographic numbering. The seven His residues, His20, His22, His31, His32, His44, His68, and His85, correspond to His15, His17, His26, His27, His39, His63, and His80, respectively, of the crystallographic data for bovine cytochrome b5 (Protein Data Bank, 1CYO).
3The heme-coordination of VHA-Mb in ferrous state is found to have a bis-His (His68/His97) structure based on visible absorption spectra, resonance Raman spectra, and cyanide-binding affinity (3). On the other hand, VHA-Mb in ferric state is reported to have a His68/hydroxide structure based on resonance Raman profiles and visible absorption spectra (3). However, cyanide-binding affinity of VHA-Mb in ferric state (Kd = 2.04 mM) was much lower than those of WT-Mb (1.87 µM), VHAF-Mb (5.85 µM), or VHGF-Mb (5.65 µM), where a water or hydroxide ion was expected to bind to the ferric heme as a distal ligand (3) and was comparable to that of the VH double mutant (8.51 mM), in which the bis-His coordination structure was verified by X-ray crystallography (4). Further, the pH titration of VHA-Mb in ferric state showed a very similar profile in spectral changes with that of VH double mutant (3). These observations suggested that the distal hydroxide ligand of VHA-Mb in ferric state might be strongly stabilized by a hydrogen bond with distal His97 residue to prevent the ligand exchange with cyanide ion or, more likely, that the His97 imidazole itself might coordinate directly to the ferric heme.
4Such relatively minor perturbation in electronic absorption spectra might be comparable to those found for H93G proximal cavity mutant of myoglobin, where the proximal His has been replaced with Gly, creating a cavity which can be occupied by a variety of exogenous ligands (5, 6). Exogenously added imidazole could coordinate heme iron of H93G myoglobin as the proximal ligand, showing almost identical spectroscopic characteristics with those of wild-type myoglobin, despite of a significant rotation (
45°) of imidazole plane and a decreased distance between the heme and the proximal imidazole (5). Further, when imidazole was replaced with other small organic ligands, such as pyridine and methyl-substituted imidazoles, relatively minor changes in absorption spectra occurred (6).
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