Fourier Transform Infrared (FTIR) and Step-scan Time-resolved FTIR Spectroscopies Reveal a Unique Active Site in Cytochromecaa3 Oxidase from Thermus thermophilus

Pinakoulaki, Eftychia; Soulimane, Tewfik; Varotsis, Constantinos

doi:10.1074/jbc.m205568200

Cited by 34 publications

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“…pK a of the Tyrosine Residue-Intensity changes and frequency shifts of side chains and backbone structures have been observed in the FTIR difference spectra of heme-copper oxidases as the result of an electrochemical perturbation (oxidized minus reduced) of the metal centers at room temperature or of the perturbation induced by the photodissociation of CO bound to the heme at 80 K, at which the CO binds irreversibly to Cu B (18,19,23,24). In the latter case, the perturbation induced by the photodissociated CO exerts its main effect on the environment of the binuclear center.…”

Section: Resultsmentioning

confidence: 99%

“…In the work presented here, we continued our TRS 2 -FTIR approach with heme-copper oxidases at room temperature, and in conjunction with the assignment of 7a (CO) and ␦(COH) in the aa 3 -type oxidase from P. denitrificans, we detected the protonated and deprotonated forms of tyrosine residue(s) near the induced perturbation, i.e. the heme a 3 -Cu B binuclear center of cytochrome ba 3 from T. thermophilus (4,5,17,18,23,24). Our TRS 2 -FTIR difference spectra show that 7a (CO) and ␦(COH) in the protonated form of Tyr are located at 1247 cm Ϫ1 and in the deprotonated form at 1301 cm Ϫ1 .…”

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confidence: 99%

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Probing Protonation/Deprotonation of Tyrosine Residues in Cytochrome ba3 Oxidase from Thermus thermophilus by Time-resolved Step-scan Fourier Transform Infrared Spectroscopy

Koutsoupakis

Kolaj-Robin

Soulimane

et al. 2011

Journal of Biological Chemistry

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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 7a (CO) of the protonated form of Tyr residues at 1247 cm

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

Probing Protonation/Deprotonation of Tyrosine Residues in Cytochrome ba3 Oxidase from Thermus thermophilus by Time-resolved Step-scan Fourier Transform Infrared Spectroscopy

Koutsoupakis

Kolaj-Robin

Soulimane

et al. 2011

Journal of Biological Chemistry

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“…Difference spectra were calculated as ⌬A ϭ Ϫlog(intensity of sample/intensity of reference). The detailed experimental set-up for the time-resolved step-scan FTIR has been described previously (7,9,11). The rate constants for each phase of the decay of the Cu B 1ϩ -CO complex and for CO rebinding to heme a 3 were calculated, assuming first-order kinetics, with three-parameter exponential fits to the experimental data.…”

Section: Methodsmentioning

confidence: 99%

“…Because of the unusual ligand-binding and kinetic properties of the binuclear center, cytochrome ba 3 oxidase is unique among the heme-copper oxidases in that it is susceptible to a detailed kinetic analysis of its ligand dynamics (4,7). The binding of CO to the binuclear center of ba 3 follows that found in all heme-copper oxidases and proceeds according to the Scheme 1 (7)(8)(9)(10)(11)(12).…”

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“…Because it has been proposed that 1) Cu B is a way-stop for ligand entry to heme a 3 (8) and 2) the O 2 channel is located at the Cu B site, we sought to determine the properties of the binuclear center by applying our FTIR approach (7,9,11) to study the CO bound cytochrome ba 3 at room temperature in the presence of low-oxygen concentrations in the medium (ϳ70 M), preventing spontaneous replacement of CO by O 2 . In cytochrome ba 3 , the exceptionally high affinity for CO binding to Cu B (K 1 Ͼ 10 4 ) has allowed us to perform such experiments.…”

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Oxygen-linked Equilibrium CuB-CO Species in Cytochrome ba3 Oxidase from Thermus thermophilus

Koutsoupakis

Stavrakis

Soulimane

et al. 2003

Journal of Biological Chemistry

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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 Cytochrome ba 3 from Thermus thermophilus is a member of the large family of structurally related heme-copper oxidases (1, 2). It catalyzes both the four-electron reduction of O 2 to H 2 O, converting the energy of this reaction to a transmembrane proton motive force, and the two-electron reduction of NO to N 2 O (1-4). Based on the crystal structure, the enzyme contains a homodinuclear copper (Cu A ), a low-spin heme b, and a heme a 3 -Cu B binuclear center (1). The ba 3 -oxidase retains the electron transport chain functional under low oxygen concentration in the medium. In both structurally characterized aa 3 -type heme-copper oxidases, three possible O 2 channels have been suggested (5, 6). One of them leads from a trapped lipid pocket in subunit III to the active site and contains Glu-278 (Paracoccus denitrificans numbering), a residue near Cu B (5, 6). In the presence of three O 2 channels in aa 3 -type oxidases, it is not known yet whether a facilitated oxygen channel is necessary, because the O 2 concentrations normally far exceed the O 2 affinity of the enzyme; thus, excess O 2 would not be rate-limiting. The proposed oxygen input channel in ba 3 contains Ile-235 instead of Glu-278 (conserved to aa 3 oxidases), which optimizes the formation of a hydrophobic pore (1, 2). The evolutionary development of an optimized oxygen channel is appropriate for organisms such as T. thermophilus ba 3 oxidase, the archaeal Sulfolobus acidocaldarius aa 3 -quinol oxidase, and Natronomonas pharaonics, which grow under low oxygen tension and at high temperatures (2). However, no data exist in the literature to demonstrate the nature of the conformational changes that occur in the binuclear center in the presence of O 2 in the channel. Because of the unusual ligand-binding and kinetic properties of the binuclear center, cytochrome ba 3 oxidase is unique among the heme-copper oxidases in that it is susceptible to a detailed kinetic analysis of its ligand dynamics (4, 7). The binding of CO to the binuclear center of ba 3 follows that found in all heme-copper oxidases and proceeds according to the Scheme 1 (7-12).In our previous work (7), we identified the C-O stretching mode of the equilibrium Cu B 1ϩ -CO species (complex A) at 2053 cm Ϫ1 and concluded that the environment in the binuclear center does not alter the protonation state of the Cu B histidine ligands. Understanding the conformational transitions that are associated with protonation/deprotonation of labile residues is essential because ionizable groups whose pK a values are near physiological pH are involved in proton uptake or release. A hydrogen-bonded connectivity between the propionates of heme a 3 , Asp-372, and H 2 O was also reported. Accordingly, plausible mechanisms of proton pathway(s) directly assoc...

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Fourier Transform-Infrared Spectroscopy for Biophysical Applications: Technical Aspects

Fahmy¹

2018

Encyclopedia of Biophysics

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Fourier Transform Infrared (FTIR) and Step-scan Time-resolved FTIR Spectroscopies Reveal a Unique Active Site in Cytochromecaa3 Oxidase from Thermus thermophilus

Abstract: Fourier transform infrared (FTIR

Cited by 34 publications

References 35 publications

Probing Protonation/Deprotonation of Tyrosine Residues in Cytochrome ba3 Oxidase from Thermus thermophilus by Time-resolved Step-scan Fourier Transform Infrared Spectroscopy

Probing Protonation/Deprotonation of Tyrosine Residues in Cytochrome ba3 Oxidase from Thermus thermophilus by Time-resolved Step-scan Fourier Transform Infrared Spectroscopy

Oxygen-linked Equilibrium CuB-CO Species in Cytochrome ba3 Oxidase from Thermus thermophilus

Fourier Transform-Infrared Spectroscopy for Biophysical Applications: Technical Aspects

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