Raman spectroscopic and electrochemical characterization of myoglobin thinfilm: Implication of the role of histidine 64 for fast heterogeneous electron transfer

Myoglobin (Mb) thin films formed on various substrates have been characteri zed by using Raman spectroscopy, reflectance absorbance FT-IR, UV-vis absor ption spectroscopy, and electrochemical methods. Raman spectra were obtaine d upon excitation within the Soret band as well as alpha-beta bands. The sp in state marker bands observed from the Mb film in the 1550-1630 cm(-1) reg ion (excitation at 514.5 nm) are similar to 20 cm(-1) higher than those of aqueous metMb having the high spin state. The 1210 cm(-1) band (methine bri dge C-H vibration) also shifts to 1240 cm(-1) upon the formation of the fil m. These results indicate that the heme iron of myoglobin in the film is th e ferric low-spin state, and the iron atom is pulled to the heme plane. A c omparison of the Raman spectra of the Mb film with that of an Mb-imidazole derivative leads to the conclusion that the distal histidine is responsible for the change in the spectral characteristics. The escape of water from t he sixth position upon the formation of the Mb film may result in a conform ational change at the heme distal pocket: the histidine residue at the E7 h elical position (H64) moves toward the central iron and is coordinated with it through the N on the imidazole ring. These structural features facilita te the fast electron transfer between the thin protein film and the electro de. Distal histidine may serve as an electron-transfer pathway as it does i n cytochrome c.