Spectral Analysis Is Suitable To Decompose Overcrowded Resonance Raman Spectra of Metalloporphyrins and Yields Reliable Depolarization Ratios

Abstract

Spiro and co-workers' have measured and compared the complex Raman spectra of the heme group embedded in yeast iso-1-cytochrome c with some of its isotopomers. Their findings lead them to the conclusion that most of the particular strong DPR dispersions earlier observed in our laboratory for the Raman lines v4, v19, and v21 of horse heart ferrocytochrome c2 result from spectral overlap with adjacent lines rather than from symmetry lowering perturbations. To check the reliability of the reported DPR values, we have reinvestigated the polarization properties of the above Raman lines by measuring polarized Raman spectra of ferrocytochrome c at various excitation wavelengths with comparatively high spectral resolution (i.e., 1.9-2.4 ~m -' ) .~ Thus, we reaffiied the earlier reported DPR dispersion for v21 (at 1312 cm-l) and v4 (at 1360 cm-l), whereas the corresponding DPR dispersion of the very intense ~1 9 band (at 1583 cm-l) was found to result in part from an overlap with the adjacent v2 mode. Moverover, it was shown that other Raman lines (Le., v2, v20) also show significant dispersion of their DPR.Hu and Spiro have now published a reply" in which they claim that the spectral crowding in the Raman spectra of ferrocytochrome is too intense to allow a reliable decomposition into single Raman lines. To support their argument for the case of the Raman line 191, they have measured the polarized spectra of yeast iso-1-ferrocytochrome c and its 2,4-di(a-dl)-isotopomer (Le., deuteration of the CH groups attached at the thioether bonds) between 1200 and 1350 cm-l at 11 K with three different excitation wavelengths. Indeed, the spectra of the natural abundance (n.a.) sample taken at 413 nm (B excitation) show that the v21 band overlaps with at least two lines at 1302 and 1317 cm-l. They are absent in the corresponding spectra of the isotopomer and are therefore assigned to the C,H bending modes of the thioether bridges. The DPR value of the isolated 1 9 1 of the above isotopomer is close to 3 (not 5 , as mentioned in ref 4) at 413, 521, and 531 nm. From this observation the authors conclude that the DPR of v21 does not show any significant dispersion.Spiro and c o -w~r k e r s~,~ have performed their measurements at 11 K to obtain a slightly better separation of overlapping bands. It is normally very difficult to obtain correct DPR values from heme proteins in an aqueous solution under this condition, because the scattered light is partially depolarized by multiple scattering in the ice even if one uses a backscattering geometry. This reduces the apparent DPR dispersion signficantly and makes comparison with our data difficult.In order to check the reliability of our previous analysis of the v21 and other relevant Raman bands, we have subjected our spectra to a new line shape analysis using the program MULTIFIT recently developed in our laboratory, which allows better and faster fitting of experimental spectra than the previously used LAB CALC. The natural Lorentzians of the Raman lines have now been convoluted with Gauss...

Enhanced Resolution of Hemoglobin Dynamics Provided by Subunit-Specific Resonance Raman Signals

“…(a) Rousseau, D. L.; Friedman, J. M. Biological Applications of Raman Spectroscopy ; In ref , Chapter 4.…”

“…The reaction coordinate between these states remains largely undefined due to the high degree of cooperativity which ensures that the intermediate states are very sparsely populated at equilibrium and accessible only by kinetic methods or by studying chemically modified derivatives which mimic the partially ligated tetramer . Resonance Raman (RR) and time-resolved resonance Raman (TR 3 ) spectroscopy − provide the capability of monitoring the detailed structure and dynamics (TR 3 ) of various molecular fragments throughout the Hb tetramer. However, all previous TR 3 studies of the photoproduct at 10 ns of native Hb were plagued by the interpretational ambiguity which arises from the fact that the observed shifts are actually an average shift; i.e., a conglomerate of signals arising from the four individual hemes present in the tetramer.…”

“…The RR spectra of deoxy-Hb and deoxy Hb-d4 (Figure ) exhibit a number of structure sensitive “marker” modes, labeled ν 10 (dp), ν 11 (dp), ν 19 (ap), and ν 3 (p),7c some of which are sensitive to deuteration (ν 10 and ν 19 ). It has been previously shown − ,, that, upon photolysis of Hb(CO) 4 with ∼10 ns laser pulses, a fully deligated initial photoproduct (Hb)* is formed whose transient RR spectrum exhibits vibrational frequencies which are slightly downshifted relative to those of the equilibrium T-state deoxy derivative. Dasgupta and Spiro 7c have carefully documented the precise magnitudes of these frequency shifts (Δ) between Hb and Hb* and have shown that the frequency of ν 19 for Hb is 3 cm -1 higher than for ν 19 of Hb*.…”

“…There have been reports on the effects of depolarization ration dispersion which can, in principle, affect peak frequencies. Such effects are generally considered to be insignificant for the types of measurements that were done here. 7c-e Spectra ( I ⊥ − I ∥ ) of both the equilibrium deoxy and photolyzed forms for both hybrids are given in Figure . Comparison of the frequencies of the ν 19 modes associated with the α h subunits of the (α h β d ) 2 hybrids (∼1550 cm -1 ) yields a 2−3 cm -1 downshift in the spectrum of (α h β d ) 2 *.…”