Myoglobin (Mb) thin films formed on various substrates have been characterized by using Raman spectroscopy, reflectance absorbance FT-IR, UV-vis absorption spectroscopy, and electrochemical methods. Raman spectra were obtained upon excitation within the Soret band as well as alpha-beta bands. The spin state marker bands observed from the Mb film in the 1550-1630 cm(-)(1) region (excitation at 514.5 nm) are approximately 20 cm(-)(1) higher than those of aqueous metMb having the high spin state. The 1210 cm(-)(1) band (methine bridge C-H vibration) also shifts to 1240 cm(-)(1) upon the formation of the film. These results indicate that the heme iron of myoglobin in the film is the ferric low-spin state, and the iron atom is pulled to the heme plane. A comparison 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 the sixth position upon the formation of the Mb film may result in a conformational change at the heme distal pocket: the histidine residue at the E7 helical position (H64) moves toward the central iron and is coordinated with it through the N on the imidazole ring. These structural features facilitate the fast electron transfer between the thin protein film and the electrode. Distal histidine may serve as an electron-transfer pathway as it does in cytochrome c.