tailieunhanh - Báo cáo Y học: Electrochemical, FT-IR and UV/VIS spectroscopic properties of the caa3 oxidase from T. thermophilus

Thecaa3-oxidase from Thermus thermophilushas been studied with a combined electrochemical, UV/VIS and Fourier-transform infrared (FT-IR) spectroscopic approach. In this oxidase the electron donor, cytochromec, 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 ) ± V (vs. Ag/AgCl/3MKCl, V vs. SHE¢) for the hemec. | Eur. J. Biochem. 269 4830-4838 2002 FEBS 2002 doi Electrochemical FT-IR and UV VIS spectroscopic properties of the caa3 oxidase from T. thermophilus Petra Hellwig1 Tewfik Soulimane2 and Werner Mantele1 1Institut fur Biophysik der Johann-Wolfgang-Goethe-Universitat Frankfurt M. Germany 2Institut fur Biochemie der Rheinisch-Westfalischen-Technischen Hochschule Aachen Germany 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 V vs. Ag AgCl 3M KCl 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 V V Em2 V V and Em3 V V and V vs. SHE respectively could be obtained. Potential titrations after addition of the inhibitor cyanide yielded a midpoint potential of V V for heme a3-CN- and of Em2 V V and Em3 V V for heme a V V and 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 cyto chrome 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 en yme to identify alternative protonatable sites. The vibrational modes of a protonated aspartic or glutamic acid at .