The origin of the FeIV=O intermediates in cytochrome aa3 oxidase

AbstractThe dioxygen reduction mechanism in cytochrome oxidases relies on proton control of the electron transfer events that drive the process. Proton delivery and proton channels in the protein that are relevant to substrate reduction and proton pumping are considered, and the current status of this area is summarized. We propose a mechanism in which the coupling of the oxygen reduction chemistry to proton translocation (P→F transition) is related to the properties of two groups of highly conserved residues, namely, His411/G386-T389 and the heme a3–propionateA–D399–H403 chain. This article is part of a Special Issue entitled: Respiratory Oxidases

Binding and Docking Interactions of NO, CO and O2 in Heme Proteins as Probed by Density Functional Theory

Dynamics and reactivity in heme proteins include direct and indirect interactions of the ligands/substrates like CO, NO and O2 with the environment. Direct electrostatic interactions result from amino acid side chains in the inner cavities and/or metal coordination in the active site, whereas indirect interactions result by ligands in the same coordination sphere. Interactions play a crucial role in stabilizing transition states in catalysis or altering ligation chemistry. We have probed, by Density Functional Theory (DFT), the perturbation degree in the stretching vibrational frequencies of CO, NO and O2 molecules in the presence of electrostatic interactions or hydrogen bonds, under conditions simulating the inner cavities. Moreover, we have studied the vibrational characteristics of the heme bound form of the CO and NO ligands by altering the chemistry of the proximal to the heme ligand. CO, NO and O2 molecules are highly polarizable exerting vibrational shifts up to 80, 200 and 120 cm−1, respectively, compared to the non-interacting ligand. The importance of Density Functional Theory (DFT) methodology in the investigation of the heme-ligand-protein interactions is also addressed

Oxygen-linked equilibrium Cu B-CO species in cytochrome ba 3 oxidase from Thermus thermophilus: implications for an oxygen channel at the Cu B site

We report the first study of O 2 migration in the putative O 2 channel of cytochrome ba 3 and its effect to the properties of the binuclear heme a 3-Cu B center of cytochrome ba 3 from Thermus thermophilus. The Fourier transform infrared spectra of the ba 3-CO complex demonstrate that in the presence of 60-80 μM O 2, the ν(C-O) of Cu B 1+-C-O at 2053 cm -1 (complex A) shifts to 2045 cm -1 and remains unchanged in H 2O/D 2O exchanges and in the pH 6.5-9.0 range. The frequencies but not the intensities of the C-O stretching modes of heme a 3-CO (complex B), however, remain unchanged. The change in the ν(C-O) of complex A results in an increase of k -2, and thus in a higher affinity of Cu B for exogenous ligands. The time-resolved step-scan Fourier transform infrared difference spectra indicate that the rate of decay of the transient Cu B 1+-CO complex at pH 6.5 is 30.4 s -1 and 28.3 s -1 in the presence of O 2. Similarly, the rebinding to heme a 3 is slightly affected and occurs with k 2 = 26.3 s -1 and 24.6 s -1 in the presence of O 2. These results provide solid evidence that in cytochrome ba 3, the ligand delivery channel is located at the Cu B site, which is the ligand entry to the heme a 3 pocket. We suggest that the properties of the O 2 channel are not limited to facilitating ligand diffusion to the active site but are extended in controlling the dynamics and reactivity of the reactions of ba 3 with O 2 and N

Observation of the equilibrium Cu B-CO complex and functional implications of the transient heme a 3 propionates in cytochrome ba 3-CO from Thermus thermophilus. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR studies

We report the first evidence for the existence of the equilibrium Cu B 1+-CO species of CO-bound reduced cytochrome ba 3 from Thermus thermophilus at room temperature. The frequency of the C-O stretching mode of Cu B 1+-CO is located at 2053 cm -1 and remains unchanged in H 2O/D 2O exchanges and, between pD 5.5 and 9.7, indicating that the chemical environment does not alter the protonation state of the Cu B histidine ligands. The data and conclusions reported here are in contrast to the changes in protonation state of Cu B-His-290, reported recently (Das, T. K., Tomson, F. K., Gennis, R. B., Gordon, M., and Rousseau, D. L. (2001) Biophys. J. 80, 2039-2045 and Das, T. P., Gomes, C. M., Teixeira, M., and Rousseau, D. L. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9591-9596). The time-resolved step-scan FTIR difference spectra indicate that the rate of decay of the transient CU B 1+-CO complex is 34.5 s -1 and rebinding to heme a 3 occurs with k 2 = 28.6 s -1. The rate of decay of the transient Cu B 1+-CO complex displays a similar time constant as the absorption changes at 1694(+)/1706(-), attributed to perturbation of the heme a 3 propionates (COOH). The ν(C-O) of the transient Cu B 1+-CO species is the same as that of the equilibrium Cu B 1+-CO species and remains unchanged in the pD range 5.5-9.7 indicating that no structural change takes place at Cu B between these states. The implications of these results with respect to proton pathways in heme-copper oxidases are discusse

Discrete Ligand Binding and Electron Transfer Properties of ba3-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 ba3 oxidase is a B-type oxidase from the extremely thermophilic eubacterium Thermus thermophilus with high O2 affinity, expressed under elevated temperatures and limited oxygen supply and possessing discrete structural, ligand binding, and electron transfer properties. The origin and the cause of the peculiar, as compared to other C cOs, thermodynamic and kinetic properties remain unknown. Fourier transform infrared (FTIR) and time-resolved step-scan FTIR (TRS2-FTIR) spectroscopies have been employed to investigate the origin of the binding and electron transfer properties of cytochrome ba3 oxidase in both the fully reduced (FR) and mixed valence (MV) forms. Several independent and not easily separated factors leading to increased thermostability and high O2 affinity have been determined. These include (i) the increased hydrophobicity of the active center, (ii) the existence of a ligand input channel, (iii) the high affinity of CuB for exogenous ligands, (iv) the optimized electron transfer (ET) pathways, (v) the effective proton-input channel and water-exit pathway as well the proton-loading/exit sites, (vi) the specifically engineered protein structure, and (vii) the subtle thermodynamic and kinetic regulation. We correlate the unique ligand binding and electron transfer properties of cytochrome ba3 oxidase with the existence of an adaption mechanism which is necessary for efficient function. These results suggest that a cascade of structural factors have been optimized by evolution, through protein architecture, to ensure the conversion of cytochrome ba3 oxidase into a high O2-affinity enzyme that functions effectively in its extreme native environment. The present results show that ba3-cytochrome c oxidase uses a unique structural pattern of energy conversion that has taken into account all the extreme environmental factors that affect the function of the enzyme and is assembled in such a way that its exclusive functions are secured. Based on the available data of CcOs, we propose possible factors including the rigidity and nonpolar hydrophobic interactions that contribute to the behavior observed in cytochrome ba3 oxidase

Antimalarial endoperoxides: synthesis and implications of the mode of action

Abstract 6,7-Dioxabicyclo[3.2.2]non-8-ene 2 and 1-isopropyl-4-methyl-2,3- dioxabicyclo[2.2.2]oct-5-ene (ascaridol) 3 were prepared as simplified, endoperoxide versions of clinically used antimalarial drugs. Fourier transform infrared (FTIR) technique in conjunction with 18O 2- enriched compound 2 has been applied in probing the bonds of the endoperoxide moiety and the bonds of the rings owing to the presence of the O-O, the C-O, the O-O-C as well as the C=O modes in the spectrum. The endoperoxide moiety is especially useful in this regard because the homolytic cleavage of the O-O bond can be characterized and hence can be used to assess the vibrational properties of the O- and C-centered radicals and subsequently that of the C-C bond cleavage. The cleavage of the O-O bond, and the ability to correlate vibrational properties of the reaction products with structural properties of the isolated products suggest that infrared spectroscopy is an appropriate tool to study the mode of action of antimalarial endoperoxides

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 stepscan Fourier transform infrared difference spectra of photodissociated CO from heme a 3 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 a3propionate. Two trajectories of CO that are distinguished spectroscopically and kinetically (Vco = 2131 cm-1, td = 10-35 μs and vco= 2146 cm -1, td = 85 μs) are observed. At later times (t d = 110 μs) the docking site reorganizes about the CO and quickly establishes an energetic barrier that facilitates equilibration of the ligand with the protein solvent. The time-dependent shift of the CO trajectories we observe is attributed to a conformational motion of the docking site surrounding the ligand. The implications of these results with respect to the ability of the docking site to constrain ligand orientation and the reaction dynamics of the docking site are discussed herei

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. The activation of this mechanism by ba3-oxidase under visible excitation raises the question as to whether such a mechanism is physiologically relevant to the extreme environment in which it operates