Characterization of heme-coordinating histidyl residues of an engineered six-coordinated myoglobin mutant based on the reactivity with diethylpyrocarbonate, mass spectrometry, and electron paramagnetic resonance spectroscopy.
A genetically engineered porcine myoglobin triple mutant (H64V/V68H/H93A) (VHA-Mb) contains 6 non-axial His residues (His24, His36, His48, His81, His82, and His119) besides two candidate axial His residues (His68 and His97). Although previous resonance Raman study on the ferric VHA-Mb were not conclusive for its coordination structure, present EPR parameters of the ferric VHA-Mb were consistent with bis-imidazole coordination of His68/His97. We further investigated the reactivity of these possible His ligands with diethylpyrocarbonate (DEPC) to clarify the coordination structure and their protonation states in ferric form. We found that the non-axial His residues were easily modified with a low concentration of DEPC based on UV spectral changes and MALDI-TOF-MS analyses. On the other hand, the two candidate axial His ligands were protected from the modification due to a limited steric exposure of their imidazoles to solvent, the Fe(3+)-N(epsilon2) coordination bond, and the protonation of N(delta1) by forming a hydrogen bond with their immediate surroundings. However, once N-carbethoxylation occurred at N(epsilon2) of His97, resulting in a disruption of the heme Fe(3+)-N(epsilon2) coordination bond, it facilitated the second N-carbethoxylation to take place at N(delta1) of the same imidazole ring, leading to a bis-N-carbethoxylated derivative and further to a ring-opened derivative. These phenomena were consistent with the bis-His68/His97 coordination. Further, these were not observed at all for cytochrome b(561), a transmembrane di-heme containing protein responsible for the ascorbate-specific transmembrane electron transfer, where only a specific N(delta1)-carbethoxylation of axial His occurred at a low concentration of DEPC, leading to an inhibition of the electron acceptance from ascorbate without a release of the heme. These distinct results might be related to a specific physiological mechanism being operative at the cytosolic heme center of cytochrome b(561).
Nakanishi
Nobuyuki
N
Department of Molecular Science and Material Engineering, Graduate School of Science and Technology, Rokkodai-cho, Nada-ku, Kobe, Hyogo 657-8501, Japan.
Takeuchi
Fusako
F
Park
Sam-Yong
SY
Hori
Hiroshi
H
Kiyota
Kohei
K
Uno
Tadayuki
T
Tsubaki
Motonari
M
D004047:Diethyl Pyrocarbonate / Q000737:chemistry*; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods*; D006418:Heme / Q000235:genetics*; D006639:Histidine / Q000235:genetics*; D013058:Mass Spectrometry / Q000379:methods*; D009154:Mutation / Q000235:genetics; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics*; D015202:Protein Engineering / Q000379:methods*; D017434:Protein Structure, Tertiary
D009211:Myoglobin; D006418:Heme; D006639:Histidine; D004047:Diethyl Pyrocarbonate