COMPLETE ASSIGNMENT OF CYTOCHROME-C RESONANCE RAMAN-SPECTRA VIA ENZYMATIC RECONSTITUTION WITH ISOTOPICALLY LABELED HEMES

Resonance Raman (RR) spectra are reported for ferrous yeast iso-1 cytochrome c and its meso-d4, pyrrole-N-15, 2,4-di(a-d1), and 2,4-di(b-d2) isotopomers, obtained by the enzymatic coupling of apoprotein with labeled hemes. Three excitation wavelengths, in resonance with the B (413.1 nm) and Q (520.8 and 530.9 nm) states, were used to selectively examine vibrational modes of different symmetry. On the basis of the observed isotope shifts and the measured depolarization ratios and by analogy to those of nickel octaethylporphyrin, RR bands are assigned to nearly all of the porphyrin in-plane and many of the out-of-plane skeletal modes. Assignments of the 2,4-thioether substituent modes are secured from their isotope shifts upon selective deuteration; the C-S stretching bands of these substituents are among the strongest bands in the B-resonant spectrum. Candidate bands are also found for modes of the methyl and propionate substituent groups. The characteristic four sets of doublet bands, clustered in the spectral region 330-430 cm-1, are assigned to the porphyrin modes nu8 and nu50 and to pairs of deformation modes of the 2,4-thioether substituents and 6,7-propionate substituents. Out-of-plane distortions of the porphyrin, seen in the high-resolution X-ray crystal structures, are manifested in the RR spectrum in the following ways: (1) two anomalously polarized bands (nu10 and nu21) gain substantial intensity in the B-state excited spectrum, and a depolarized band (nu15) is extraordinarily strong; (2) many IR-active E(u) modes become RR-active, and some of them split into doublets; (3) out-of-plane deformation modes involving methine wagging, pyrrole folding, and pyrrole swiveling coordinates are activated, and two E(g) modes (gamma20 and gamma21) are split. Band frequencies are almost the same for ferric as ferrous cytochrome c, except for the high-frequency skeletal modes, which reflect the different extent of back-bonding. The relative intensities are also very similar, reflecting the minimal change in heme structure in the two oxidation states.