Resonance Raman spectra of cytochrome a in cytochrome oxidase: Excitation in the 605‐nm absorption region

Abstract

Visible excitation resonance Raman difference spectra, in resonance with the 605-nm absorption maximum of fully reduced and mixed-valence cyanide-bound cytochrome oxidase, were recorded. Under these conditions, the vibrations of ferrocytochrome a are markedly enhanced owing to its dominant contribution to the 605nm a-band absorption of reduced cytochrome oxidase. The effect of H/D exchange in the Raman spectra of cytochrome a was also investigated as a way to establish formyl-and vinyl-peripheral substituent sensitive modes. Measurements of the depolarization ratio dependence of cytocbrome a vibrational modes resulted in some isotope-sensitive modes with characteristic depolarized behavior ; however, it was observed that the majority of the visible excitation vibrations exhibit polarized values. This is interpreted as being due to a significant lowering in the molecular symmetry of the heme a porphyrin macrocycle caused by the unusual peripheral substituent pattern of heme a and, presumably, by the strong cytochrome a-protein interactions. Both structural effects appear to result in a selective enhancement of cytocbrome a Ew substituent-sensitive modes as detected by the low-frequency resonance Raman spectrum.

Resonance Raman studies on the CuA site of cytochrome c oxidase using a multichannel scanning Raman spectrometer with a CCD detector

“…The intensities of the redox state dependent bands were relatively enhanced upon excitation at 735 nm (not shown), indicating that these bands are not in resonance with the 830nm band and accordingly are assigned to heme modes. In fact, the observed frequencies for the CO-bound fully reduced form coincide with those obtained by the -band excited resonance Raman spectra (Bocian et al, 1979;Callahan & Babcock, 1983;Centeno & Babcock, 1991).…”

“…The CuA-ligand stretching [see Woodruff et al (1988) for a review]. Excitation of Raman scattering from blue copper proteins by red light around 590-610 nm yielded a number of RR bands below 500 cm-1 assignable to the Cu-Cys and Cu-His stretching and related modes (Han et al, 1991), but red excitations of cytochrome c oxidase brought no CuA-associated Raman bands (Bocian et al, 1979;Callahan & Babcock, 1983;Centeno & Babcock, 1991). This failure has been probably caused by the low sensitivity of a photomultiplier in the far-red region.…”

Comparative resonance Raman study of cytochrome c oxidase from beef heart and Paracoccus denitrificans

“…Vibrational Assignments of the Reduced Enzymes. Most of the band assignments for the RR spectrum of BOXred are generally agreed upon, on the basis of the body of data obtained from heme a3 ligated species, variable wavelength studies, and model compound spectra (Centeno & Babcock, 1991;Choi et al, 1983; Argade et al, 1986;Ching et al, 1985; Willems & Bocian, 1984). Hence, in particular for most of the heme a modes (v]0, ¡>2, v\ 1,1/3, and vis) including the heme a formyl stretching vibration at 1609 cm™1, we have adopted the assignments proposed previously.…”

“…We assign these band pairs to the x and y components of 1/33, respectively. For the heme a band at 1568 cm™1, Centeno and Babcock (1991) recently suggested an alternative assignment (*>37) based on the RR spectrum excited in 605-nm absorption. This, however, would imply an unusually low frequency and strong resonance enhancement upon Soret band excitation for this mode.…”

Mitochondrial malfunction and atrophy of astrocytes in the aged human cerebral cortex

“…Analysis of Raman spectra obtained with 633 nm laser: Raman spectra obtained with 633 nm excitation have the same “lipid” and “protein” peaks with maximum positions at 1440 and 1660 cm −1 , as in spectra with 532 nm excitation, and the broad peak in the region 1550–1580 cm −1 corresponding to vibrations of different heme bonds in reduced a-type cytochromes 51 . To estimate the relative amount of reduced a-type cytochromes, we used the ratio of the sum of intensities in the regions 1550–1580 cm −1 and 1655–1665 cm −1 ( I [1550-1580] / I [1655-I1665] ).…”