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 +/- 0.01 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 +/- 0.01 V, Em2 = 0.04 V +/- 0.01 V and Em3 = 0.17 V +/- 0.02 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 +/- 0.01 V for heme a3-CN- and of Em2 = 0.00 V +/- 0.02 V and Em3 = 0.17 V +/- 0.02 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 nu(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.
D003486:Cyanides / Q000737:chemistry; D003574:Cytochrome c Group / Q000378:metabolism; D045264:Cytochromes a; D045266:Cytochromes a3; D004563:Electrochemistry; D004579:Electron Transport; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D006418:Heme / Q000737:chemistry; D010084:Oxidation-Reduction; D011522:Protons; D013053:Spectrophotometry; D013056:Spectrophotometry, Ultraviolet; D017550:Spectroscopy, Fourier Transform Infrared; D016963:Thermus thermophilus / Q000201:enzymology*
D003486:Cyanides; D003574:Cytochrome c Group; D045266:Cytochromes a3; D011522:Protons; C435384:cytochrome caa(3); D006418:Heme; D045264:Cytochromes a; D003576:Electron Transport Complex IV