UV/Vis και FTIR φασματοσκοπική μελέτη της δέσμευσης του διοξειδίου του άνθρακα (CO2) στην κυτοχρωμική οξείδαση ba3 από το Thermus thermophilus

Abstract

Cytochrome c oxidase is the final complex of the respiratory chain and catalyzes the exergonic four-electron reduction of molecular oxygen (O2) to water (H2O). This redox-driven enzyme conserves part of the released free energy generating a proton motive force that leads to the synthesis of the main biological energy source ATP. Ba3-cytochrome c oxidase is a B-type oxidase from the extremely thermophilic eubacterium Thermus thermophilus, expressed under elevated temperatures, thus limited oxygen supply, and subjected to discrete ligand binding and electron transfer properties. The ba3 oxygen reductase from Thermus thermophilus is the only B-type oxidase whose crystallographic structure has been determined. It consists of three subunits with a total of 764 amino acid residues. The low-spin heme b and the binuclear heme a3-CuB active center are contained in subunit I and the mixed valence homodinuclear (CuA1.5+-CuA1.5+) copper complex in subunit II. In addition to the 4 e- reduction of O2, cytochrome ba3 also catalyzes the 2 e- reduction of nitric oxide (NO) to nitrous oxide (N2O), and the 2 e- oxidation of carbon monoxide (CO) to carbon dioxide (CO2), with the latter having an overall scheme: Fea33+-CuB2+ + CO + H2O  Fea32+-CuB1+ + CO2 + 2 H+ Therefore, since carbon dioxide is the product of a catalytically active path of cytochrome ba3, the characterization of its binding to the binuclear center becomes an important issue. The objective of this study is to reveal the CO2 mode of binding toba3-cytochrome c oxidase. Ultraviolet/visible (UV/Vis) and Fourier transform Infrared (FTIR)spectroscopies are structure- and redox-sensitive tools able to monitor the binding, of carbon dioxide, a catalytically active ligand. Carbon dioxide (CO2),through its antisymmetric stretching mode, carries a clear and strong infrared signature, that can be used as a sensitive probe of its interactions into the binuclear center. The obtained UV/Vis and FTIR spectra are compatible with a weak, non-bonding binding of carbon dioxide into a secondary binding site of the enzyme. This binding is governed by electrostatic interactions, evidenced by the small downshift of the antisymmetric stretching vibration of CO2. The combined results suggest as the binding site the previously revealed cavity next to heme a3, which has been proven as a temporary host of ligands and lead to mechanistic conclusions about the possible, thermodynamically and kinetically, active paths that other catalytically important molecules also may follow. These paths are known to play significant role during catalysis, mainly through ligand (substrate) recognition and discrimination and serve as entrance (input) and/or exit (output) channels to and/or from the active center.