| Abstract |
The aim of the present study was the spectroscopic investigation of cytochrome ba3 from Thermus Thermophillus. Cytochrome ba3 is a member of the large family of heme-copper oxidases, which appears to have many similarities with other members of the group but reveals a number of unusual features as well. The active cite of the enzyme consists of a high spin heme a3 and a CuB atom. Based on the crystal structure, ba3 contains two more redox centers: a low spin heme b and homobinuclear CuA center. Besides it’s ability to catalyze the four electron reduction of O2 to H2O and to pump protons through the inner mitochondrial membrane, it has been recently reported that ba3¬ is also capable of reducing nitric oxide (NO) to nitrous oxide (N2O), under reducing anaerobic conditions. In the present study we have applied FTIR and Time Resolved Step-Scan FTIR spectroscopy to investigate the structural and dynamic characteristics of cytochrome oxidase ba3. Due to it’s high sensitivity, spectral and time resolution FTIR spectroscopy allows the detection of transient structural changes even of an individual protein residue and is proven to be a very powerful tool in the study of biological systems. Experiments were carried out using the stable complex of the reduced enzyme with CO. The study of the equilibrium complex suggests that almost 30% of bound CO remains bound to CuB. The protein environment of CuB shows great stability to pH or pD changes, results that come in agreement with published studies. By photolysing the heme-Fe-CO complex, CO migrates to CuB, where it remains for a few milliseconds. Detection of the photolysis product in continuous photolysis spectra reveals information for the secondary structure of the protein and allows the identification of vibrational modes of protein groups that are influenced by the photolysis process. The study of the protein dynamics and the dynamics of the active center was achieved by the Time Resolved Step-Scan FTIR spectrums that confirmed the slow kinetic behavior of cytochrome ba3 compared to other members of the oxidase family. By detecting the protein reactions to CO photolysis from the heme iron, it was concluded that the transient binding of CO to CuB is dynamically linked to structural changes at the ring A propionate of heme a3 and the to changes in the local environment of Asp372. These groups belong to the proposed Q proton channel and the time evolution of the vibrational modes that are attributed to them, indicates that the participation of the protonated form of the aspartic residue terminates sooner than the participation of it’s deprotonated form and of the propionic groups. The perturbation of Asp372 is related to the formation of the transient complex and not to the ligand recombination to the iron of heme a3. The protein motions in the region of the propionic groups and the nearby Asp372 are performed in a way that alters the hydrogen bond network of these groups. Based on the spectroscopic data and the participation of these groups in a proton pathway which, terminates at an accumulation of water molecules, a model is proposed for their role in proton movement.
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FTIR και Time Resolved Step-Scan FTIR Μελέτη της Κυτοχρωμικής Οξειδάσης ba3 από τον οργανισμό Thermus Thermophillus
