Electrochemical, FT‐IR and UV/VIS spectroscopic properties of the caa3 oxidase from T. thermophilus

The caa3‐oxidase from Thermus thermophilus has been studied with a combined electrochemical, UV/VIS and Fourier‐transform infrared (FT‐IR) spectroscopic approach. In this oxidase the electron donor, cytochrome c, is covalently bound to subunit II of the cytochrome c oxidase. Oxidative electrochemical redox titrations in the visible spectral range yielded a midpoint potential of −0.01 ±â€ƒ0.01 V (vs. Ag/AgCl/3m KCl, 0.218 V vs. SHE′) for the heme c. This potential differs for about 50 mV from the midpoint potential of isolated cytochrome c, indicating the possible shifts of the cytochrome c potential when bound to cytochrome c oxidase. For the signals where the hemes a and a3 contribute, three potentials, = −0.075 V ±â€ƒ0.01 V, Em2 = 0.04 V ±â€ƒ0.01 V and Em3 = 0.17 V ±â€ƒ0.02 V (0.133, 0.248 and 0.378 V vs. SHE′, respectively) could be obtained. Potential titrations after addition of the inhibitor cyanide yielded a midpoint potential of −0.22 V ±â€ƒ0.01 V for heme a3‐CN– and of Em2 = 0.00 V ±â€ƒ0.02 V and Em3 = 0.17 V ±â€ƒ0.02 V for heme a (−0.012 V, 0.208 V and 0.378 V vs. SHE′, respectively). The three phases of the potential‐dependent development of the difference signals can be attributed to the cooperativity between the hemes a, a3 and the CuB center, showing typical behavior for cytochrome c oxidases. A stronger cooperativity of CuB is discussed to reflect the modulation of the enzyme to the different key residues involved in proton pumping. We thus studied the FT‐IR spectroscopic properties of this enzyme to identify alternative protonatable sites. The vibrational modes of a protonated aspartic or glutamic acid at 1714 cm−1 concomitant with the reduced form of the protein can be identified, a mode which is not present for other cytochrome c oxidases. Furthermore modes at positions characteristic for tyrosine vibrations have been identified. Electrochemically induced FT‐IR difference spectra after inhibition of the sample with cyanide allows assigning the formyl signals upon characteristic shifts of the ν(C=O) modes, which reflect the high degree of similarity of heme a3 to other typical heme copper oxidases. A comparison with previously studied cytochrome c oxidases is presented and on this basis the contributions of the reorganization of the polypeptide backbone, of individual amino acids and of the hemes c, a and a3 upon electron transfer to/from the redox active centers discussed.