The heme environment in barley hemoglobin

To elucidate the environment and ligand structure of the heme in barley hem oglobin (Hb), resonance Raman and electron paramagnetic resonance spectrosc opic studies have been carried out. The heme is shown to have bis-imidazole coordination, and neither of the histidines has imidazolate character. Bar ley Hb has a unique heme environment as judged from the Fe-CO and C-O stret ching frequencies in the CO complex. Two Fe-CO stretching modes are observe d with frequencies at 534 and 493 cm(-1), with relative intensities that ar e pH sensitive. The 534 cm(-1) conformer shows a deuterium shift, indicatin g that the iron-bound CO is hydrogen-bonded, presumably to the distal histi dine, A C-O stretching mode at 1924 cm(-1) is assigned as being associated with the 534 cm(-1) conformer. Evidence is presented that the high Fe-CO an d low C-O stretching frequencies (534 and 1924 cm(-1), respectively) arise from a short hydrogen bond between the distal histidine and the CO. The 493 cm(-1) conformer arises from an open conformation of the heme pocket and b ecomes the dominant population under acidic conditions when the distal hist idine moves away from the CO. Strong hydrogen bonding between the bound lig and and the distal histidine in the CO complex of barley Hb implies that a similar structure may occur in the oxy derivative, imparting a high stabili ty to the bound oxygen. This stabilization is confirmed by the dramatic dec rease in the oxygen dissociation rate compared with sperm whale myoglobin.