STRUCTURAL-CHANGES IN CYTOCHROME-C UPON HYDROGEN-DEUTERIUM EXCHANGE

The resonance Raman spectra of yeast ferri- and ferro-iso-1-cytochrome c dissolved in H2O and D2O are reported. Hydrogen exchange in the pro tein leads to distinct spectral changes of heme vibrational bands, par ticularly in the region between 670 and 710 cm-1 and at approximately 443 and approximately 450 cm-1. The latter two bands, which have previ ously been assigned to porphyrin modes including bending vibrations of the propionate side chains [Hildebrandt, P. (1991) J. Mol. Struct, 24 2, 379-395], reveal frequency shifts by up to 4 cm-1. These shifts are attributed to structural changes of the propionate groups caused by t he energetic differences of the hydrogen and deuterium bonds between t hese substituents and the adjacent amino acid residues. The frequency shifts of the bands between 670 and 710 cm-1 most likely reflect struc tural differences of the tetrapyrrole macrocycle itself. Time-dependen t experiments revealed that the hydrogen exchange processes associated with the changes in the resonance Raman spectra are complete in less than 15 min. The protons which are involved are those in the interior of the heme pocket as concluded by comparison with the exchange rate c onstants previously determined by NMR spectroscopy [Mayne, L., Paterso n, Y., Cerasoli, D., & Englander, S. W. (1992) Biochemistry 31, 10678- 10685]. These protons are part of a hydrogen bonding network including the amide protons of Asn-52, Met-80, and Lys-79, the side chain proto ns of Asn-52, Tyr-67, Thr-78, Trp-59, and Thr-49, and the water molecu les 121 and 166. The subtle alterations of the hydrogen bonding intera ctions which are induced by hydrogen-deuterium exchange are apparently sufficient to cause structural changes in the heme which are detected by the resonance Raman spectrum. The present results demonstrate that the hydrogen bonding network in the heme pocket of cytochrome c sensi tively controls the conformation of the porphyrin.