REDOX-DEPENDENT CHANGES AT THE HEME PROPIONATES IN CYTOCHROME-C-OXIDASE FROM PARACOCCUS-DENITRIFICANS - DIRECT EVIDENCE FROM FTIR DIFFERENCE SPECTROSCOPY IN COMBINATION WITH HEME PROPIONATE C-13 LABELING

Specific isotope labeling at the carboxyl groups of the four heme prop ionates of cytochrome c oxidase from Paracoccus denitrificans was used in order to assign signals observed in electrochemically induced redo x 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 replac ement with a kanamycin resistance cartridge, resulting in a stable 5-a minolevulinic acid (ALA) auxotrophy, Normal growth of this deficient s train and cytochrome c oxidase yield comparable to that of P. dentrifi cans wild-type strain PD1222 could be obtained by supplementation with 0.1 mM ALA in the growth medium. Visible spectra and reduced-minus-ox idized FTIR spectra showed that the purified cytochrome c oxidase had spectral characteristics identical to those of the wild-type enzyme. T he decrease of a negative signal at 1676 cm(-1) in the reduced-minus-o xidized FTIR difference spectra of the C-13-labeled cytochrome c oxida se in comparison to those of the unlabeled protein allowed the assignm ent of this signal to a COOH vibration mode of at least one of the fou r heme propionates. Moreover, a negative band at approximately 1570 cm (-1) shifted to smaller wavenumbers in the spectra of the C-13-labeled enzyme in comparison to the spectra of the unlabeled enzyme and was t hus 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 th e 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 o f the C-13-labeled enzyme, is due to a symmetric COO-mode from at leas t one heme propionate. These results suggest that at least two of the four heme propionates in cytochrome c oxidase undergo significant vibr ational changes upon reduction of the enzyme, either by protonation/de protonation or by environmental changes.