Discrete Ligand Binding and Electron Transfer Properties of ba-Cytochrome c Oxidase from Thermus thermophilus: Evolutionary Adaption to Low Oxygen and High Temperature Environments.

Cytochrome c oxidase (C cO) couples the oxidation of cytochrome c to the reduction of molecular oxygen to water and links these electron transfers to proton translocation. The redox-driven C cO conserves part of the released free energy generating a proton motive force that leads to the synthesis of the main biological energy source ATP. Cytochrome ba oxidase is a B-type oxidase from the extremely thermophilic eubacterium Thermus thermophilus with high O affinity, expressed under elevated temperatures and limited oxygen supply and possessing discrete structural, ligand binding, and electron transfer properties.

Photobiochemical production of carbon monoxide by Thermus thermophilus ba3 -cytochrome c oxidase.

We report the photobiochemical production of carbon monoxide by a terminal ba3 -cytochrome c oxidase from T. thermophilus HB8. FTIR and time-resolved step-scan FTIR spectroscopies were combined to probe this process and also monitor the concomitant binding of the produced gas to other intact ba3 molecules forming the ba3 -CO complex.

Spectroscopic and kinetic investigation of the fully reduced and mixed valence states of ba3-cytochrome c oxidase from Thermus thermophilus: a Fourier transform infrared (FTIR) and time-resolved step-scan FTIR study.

The complete understanding of a molecular mechanism of action requires the thermodynamic and kinetic characterization of different states and intermediates. Cytochrome c oxidase reduces O(2) to H(2)O, a reaction coupled to proton translocation across the membrane. Therefore, it is necessary to undertake a thorough characterization of the reduced form of the enzyme and the determination of the electron transfer processes and pathways between the redox-active centers.

Aldoxime dehydratase: probing the heme environment involved in the synthesis of the carbon-nitrogen triple bond.

Fourier transform infrared (FTIR) spectra, "light" minus "dark" difference FTIR spectra, and time-resolved step-scan (TRS(2)) FTIR spectra are reported for carbonmonoxy aldoxime dehydratase. Two C-O modes of heme at 1945 and 1964 cm(-1) have been identified and remained unchanged in H(2)O/D(2)O exchange and in the pH 5.6-8.

Probing protonation/deprotonation of tyrosine residues in cytochrome ba3 oxidase from Thermus thermophilus by time-resolved step-scan Fourier transform infrared spectroscopy.

Elucidating the properties of the heme Fe-Cu(B) binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the ν(7a)(CO) of the protonated form of Tyr residues at 1247 cm(-1) and that of the deprotonated form at 1301 cm(-1). By monitoring the intensity changes of the 1247 and 1301 cm(-1) modes as a function of pH, we measured a pK(a) of 7.

Time-resolved step-scan Fourier transform infrared investigation of heme-copper oxidases: implications for O2 input and H2O/H+ output channels.

We have applied FTIR and time-resolved step-scan Fourier transform infrared (TRS(2)-FTIR) spectroscopy to investigate the dynamics of the heme-Cu(B) binuclear center and the protein dynamics of mammalian aa(3), Pseudomonas stutzeri cbb(3), and caa(3) and ba(3) from Thermus thermophilus cytochrome oxidases. The implications of these results with respect to (1) the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases, and (2) the proton pathways located in the ring A propionate of heme a(3)-Asp372-H(2)O site that is conserved among all structurally known oxidases are discussed.

Probing the Q-proton pathway of ba3-cytochrome c oxidase by time-resolved Fourier transform infrared spectroscopy.

In cytochrome c oxidase, the terminal respiratory enzyme, electron transfers are strongly coupled to proton movements within the enzyme. Two proton pathways (K and D) containing water molecules and hydrophobic amino acids have been identified and suggested to be involved in the proton translocation from the mitochondrial matrix or the bacterial cytoplasm into the active site. In addition to the K and D proton pathways, a third proton pathway (Q) has been identified only in ba3-cytochrome c oxidase from Thermus thermophilus, and consists of residues that are highly conserved in all structurally known heme-copper oxidases.

Ligand binding in a docking site of cytochrome C oxidase: a time-resolved step-scan Fourier transform infrared study.

The description of reaction regulation in enzymes responsible for activating and catalyzing small molecules (O(2), NO) requires identification of ligand movement into the binding site and out of the enzyme through specific channels and docking sites. We have used time-resolved step-scan Fourier transform infrared spectroscopy on CO-photolyzed cytochrome c oxidase ba(3) from T. thermophilus, which is responsible for the activation and reduction of both O(2) and NO, to gain insight into the structure of ligand-binding intermediates at ambient temperature.

Docking site dynamics of ba3-cytochrome c oxidase from Thermus thermophilus.

Ligand trajectories trapped within a docking site or within an internal cavity near the active site of proteins are important issues toward the elucidation of the mechanism of reaction of such complex systems, in which activity requires the shuttling of oriented ligands to and from their active site. The ligand motion within ba3-cytochrome c oxidase from Thermus thermophilus has been investigated by measuring time-resolved step-scan Fourier transform infrared difference spectra of photodissociated CO from heme a3 at ambient temperature. Upon photodissociation, 15-20% of the CO is not covalently attached to CuB but is trapped within a docking site near the ring A of heme a3 propionate.