title: Protonation and hydrogen-bonding state of the distal histidine in the CO complex of horseradish peroxidase as studied by ultraviolet resonance Raman spectroscopy.
abstract: Ultraviolet resonance Raman (UVRR) spectroscopy has been used to characterize the structure and hydrogen bonding state of the distal histidine (His42) in horseradish peroxidase (HRP) complexed with carbon monoxide (HRP-CO). The HRP-CO - HRP UVRR difference spectrum in D(2)O solution at pD 7.0 shows two positive peaks at 1408 and 1388 cm(-)(1), which are ascribable to medium-to-weak and strong hydrogen bonding states, respectively, of the protonated imidazolium side chain of His42 in HRP-CO. Both His42 peaks decrease in intensity with increase of pD with a midpoint of transition at pD 8.8, indicating that the pK(a) of His42 in HRP-CO is 8.8. The CO ligand exhibits two C-O stretching Raman peaks at 1932 and 1902 cm(-)(1), the latter of which diminishes at alkaline pD and is assignable to a strong hydrogen-bonded state. It is most probable that the imidazolium side chain of His42 forms a strong hydrogen bond with CO, giving a His42 peak at 1388 cm(-)(1) and a CO peak at 1902 cm(-)(1), in one conformer. The other hydrogen bonding state of His42, giving the 1408 cm(-)(1) peak, is ascribed to another conformer forming a medium-to-weak hydrogen bond with a water molecule in the distal cavity. The present finding that His42 can act as a strong proton donor to CO and decrease the CO bond order is consistent with the role of His42 as a general acid to cleave the O-O bond of hydrogen peroxide, a specific oxidizing agent, in the catalytic cycle of HRP.
authors.affiliation: Faculty of Science and Engineering, Tokyo University of Science, Yamaguchi, Daigaku Dori, Sanyo Onoda, Yamaguchi 756-0884, Japan. shinji@ed.yama.tus.ac.jp
authors.forename: Shinji
authors.initials: S
authors.lastname: Hashimoto
authors.forename: Hideo
authors.initials: H
authors.lastname: Takeuchi
mesh_terms: D001665:Binding Sites; D002248:Carbon Monoxide / Q000737:chemistry*; D017666:Deuterium Oxide / Q000737:chemistry; D006639:Histidine / Q000737:chemistry*; D006735:Horseradish Peroxidase / Q000737:chemistry*; D006860:Hydrogen Bonding; D007093:Imidazoles / Q000737:chemistry; D011522:Protons; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman / Q000379:methods*
chemical_list: D007093:Imidazoles; D011522:Protons; D006639:Histidine; D002248:Carbon Monoxide; D006735:Horseradish Peroxidase; D017666:Deuterium Oxide; C023439:4-methylimidazole