Protonation and hydrogen-bonding state of the distal histidine in the CO complex of horseradish peroxidase as studied by ultraviolet resonance Raman spectroscopy.

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.