SECONDARY AND TERTIARY STRUCTURE OF THE A-STATE OF CYTOCHROME-C FROM RESONANCE RAMAN-SPECTROSCOPY

Ferricytochrome c can be converted to the partially folded A-state at pH 2.2 in the presence of 1.5 M NaCl. The structure of the A-state has been studied in comparison with the native and unfolded states, using resonance Raman spectroscopy with visible and ultraviolet excitation wavelengths. Spectra obtained with 200 nm excitation show a decrease i n amide II intensity consistent with loss of structure for the 50s and 70s helices. The 230-nm spectra contain information on vibrational mo des of the single Trp 59 side chain and the four tyrosine side chains (Tyr 48, 67, 74, and 97). The Trp 59 modes indicate that the side chai n remains in a hydrophobic environment but loses its tertiary hydrogen bond and is rotationally disordered. The tyrosine modes Y8b and Y9a s how dis ruption of tertiary hydrogen bonding for the Tyr 48, 67, and 7 4 side chains. The high-wavenumber region of the 406.7-nm resonance Ra man spectrum reveals a mixed spin heme iron atom, which arises from ax ial coordination to His 18 and a water molecule. The low-frequency spe ctral region reports on heme distortions and indicates a reduced degre e of interaction between the heme and the polypeptide chain. A structu ral model for the A-state is proposed in which a folded protein subdom ain, consisting of the heme and the N-terminal, C-terminal, and 60s he lices, is stabilized through nonbonding interactions between helices a nd with the heme.