Datensatz

Zusammenfassung

Specific isotope labeling at the carboxyl groups of the four heme propionates of cytochrome c oxidase from Paracoccus denitrificans was used in order to assign signals observed in electrochemically induced redox Fourier transform infrared (FTIR) difference spectra of this enzyme. For this purpose, the hemA gene of the P. denitrificans strain PD1222, coding for 5-aminolevulinate synthase, was deleted by partial replacement with a kanamycin resistance cartridge, resulting in a stable 5-aminolevulinic acid (ALA) auxotrophy. Normal growth of this deficient strain and cytochrome c oxidase yield comparable to that of P. dentrificans wild-type strain PD1222 could be obtained by supplementation with 0.1 mM ALA in the growth medium. Visible spectra and reduced-minus-oxidized FTIR spectra showed that the purified cytochrome c oxidase had spectral characteristics identical to those of the wild-type enzyme. The decrease of a negative signal at 1676 cm^-1 in the reduced-minus-oxidized FTIR difference spectra of the ¹³C-labeled cytochrome c oxidase in comparison to those of the unlabeled protein allowed the assignment of this signal to a COOH vibration mode of at least one of the four heme propionates. Moreover, a negative band at approximately 1570 cm^-1 shifted to smaller wavenumbers in the spectra of the ¹³C-labeled enzyme in comparison to the spectra of the unlabeled enzyme and was thus assigned to contributions from an antisymmetric COO^- mode of one or more of the four heme propionates. Additionally, a positive signal at 1538 cm^-1 shifted to approximately 1500 cm^-1 in the spectra of the isotopically labeled protein and was therefore assigned to at least one antisymmetric COO^- mode of the heme propionates. A negative signal at 1390 cm^-1, which has been shifted to 1360 cm-1 in the spectra of the ¹³C-labeled enzyme, is due to a symmetric COO^- mode from at least one heme propionate. These results suggest that at least two of the four heme propionates in cytochrome c oxidase undergo significant vibrational changes upon reduction of the enzyme, either by protonation/deprotonation or by environmental changes.