UV resonance Raman probe of heme-bound imidazole established by N-15-labeling of hemoglobin

Recent studies of Cu, Zn superoxide dismutase, and of zinc-finger peptides have established that histidine ligands can be detected in ultraviolet reso nance Raman (UVRR) spectra, following NH/D exchange of the imidazole. UVRR spectroscopy therefore offers promise for monitoring histidine Ligation in heme proteins. In this work, we characterize heme-bound histidine UVRR band s for N-acetyl-microperoxidase-8 (MP-8) and microperoxidase-11 (MP-11), and also for hemoglobin (fib). The Hb UVRR spectra are dominated by tyrosine a nd tryptophan contributions, but a band appears at 1340 cm(-1) in D2O solut ion, which is assigned to a mode of Fe-bound imidazole. This band shifted 2 4 cm(-1) in protein which was labeled with N-15 via expression of the Hb ge ne in E. coli grown on (NH4+)-N-15. In MP-11, the position of this band is insensitive to ligation or oxidation state changes, but it is 2 cm(-1) lowe r in deoxyHb than in the CO adduct. This shift may reflect mechanical force s on the proximal histidine in the T state, and/or changes in its H-bonding .