Water molecule reorganization in cytochrome c oxidase revealed by FTIR spectroscopy

Abstract

Although internal electron transfer and oxygen reduction chemistry in cytochrome c oxidase are fairly well understood, the associated groups and pathways that couple these processes to gated proton translocation across the membrane remain unclear. Several possible pathways have been identified from crystallographic structural models; these involve hydrophilic residues in combination with structured waters that might reorganize to form transient proton transfer pathways during the catalytic cycle. To date, however, comparisons of atomic structures of different oxidases in different redox or ligation states have not provided a consistent answer as to which pathways are operative or the details of their dynamic changes during catalysis. In order to provide an experimental means to address this issue, FTIR spectroscopy in the 3,560-3,800 cm(-1) range has been used to detect weakly H-bonded water molecules in bovine cytochrome c oxidase that might change during catalysis. Full redox spectra exhibited at least four signals at 3,674(+), 3,638(+), 3,620(-), and 3,607(+) cm(-1). A more complex set of signals was observed in spectra of photolysis of the ferrous-CO compound, a reaction that mimics the catalytic oxygen binding step, and their D(2)O and H(2)(18)O sensitivities confirmed that they arose from water molecule rearrangements. Fitting with Gaussian components indicated the involvement of up to eight waters in the photolysis transition. Similar signals were also observed in photolysis spectra of the ferrous-CO compound of bacterial CcO from Paracoccus denitrificans. Such water changes are discussed in relation to roles in hydrophilic channels and proton/electron coupling mechanism.

Fig. 1.

Electrochemically induced reduced minus oxidized ATR-FTIR difference spectra of bovine CcO. (A) High-frequency (3,875–3,560 cm^-1) region (black). Data were fitted with a combination of four Gaussian components, each of 7–9 cm^-1 FWHM (red trace; individual Gaussians shown in green); (B) 1,800–1,400 cm^-1 region.

Fig. 2.

Comparison of light-induced CO-photolysis transmission FTIR difference spectra of fully reduced CcO-CO from bovine and P. denitrificans CcOs. (A) Bovine CcO: FTIR difference spectra (black traces) were recorded in H₂¹⁶O (top trace, average of 4,000 transitions), D₂O (middle trace, average of 984), and H₂¹⁸O (lower trace, average of 1,500) media. Data in H₂¹⁶O and H₂¹⁸O media were fitted with combinations of Gaussian components all with a fixed FWHM of 6 cm^-1 (red traces; individual Gaussians shown in green). Fitted band frequencies are tabulated in Table S1; (B) P. denitrificans CcO: FTIR difference spectrum (black trace) in H₂¹⁶O at pH 8.5 (average of 1,400 transitions). Data were fitted with combinations of Gaussian components all with a fixed FWHM of 6 cm^-1 (red traces; individual Gaussians shown in green).

Fig. 3.

Comparison of the hydrophilic channels and their associated water molecules in bovine and P. denitrificans CcOs. Crystal structures of subunit I of oxidized CcOs from bovine (PDB ID code 1V54 (26), green) and P. denitrificans (PDB ID code 3HB3 (13), orange) were aligned on their a₃ hemes. Amino acids and crystallographically resolved water molecules within the D, K, and H channels are displayed as spheres (green, bovine CcO; orange, P. denitrificans CcO). See text for details.