Kinetic Properties of ba3 Oxidase from Thermus thermophilus:  Effect of Temperature

Giuffrè, Alessandro; Forte, Elena; Antonini, Giovanni; D’Itri, Emilio; Brunori, Maurizio; Soulimane, Tewfik; Buse, Gerhard

doi:10.1021/bi9815389

Cited by 76 publications

(115 citation statements)

References 41 publications

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“…This feature is not observed in the NO-bound form, and, by analysis in comparison with the CO second-derivative spectra (21), we estimate that NObinding occurs for more than 97%. This finding is consistent with the observation that NO complements ligand binding to heme a 3 in the fraction of CO-equilibrated enzymes that does not form the heme a 3 -CO complex (24).…”

Section: Resultssupporting

confidence: 91%

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NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa₃ from Paracoccus denitrificans and ba₃ from Thermus thermophilus

et al. 2004

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Cytochrome c oxidase (CcO) has a high affinity for nitric oxide (NO), a property involved in the regulation of respiration. It has been shown that the recombination kinetics of photolyzed NO with reduced CcO from Paracoccus denitrificans on the picosecond time scale depend strongly on the NO/ enzyme stoichiometry and inferred that more than one NO can be accommodated by the active site, already at mildly suprastoichiometric NO concentrations. We have largely extended these studies by monitoring rebinding dynamics from the picosecond to the microsecond time scale, by performing parallel steadystate low-temperature electron paramagnetic resonance (EPR) characterizations on samples prepared similarly as for the optical experiments and comparing them with molecular-modeling results. A comparative study was performed on CcO ba 3 from Thermus thermophilus, where two NO molecules cannot be copresent in the active site in the steady state because of its NO reductase activity. The kinetic results allow discrimination between different models of NO-dependent recombination and show that the overall NO escape probability out of the protein is high when only one NO is bound to CcO aa 3 , whereas strong rebinding on the 15-ns time scale was observed for CcO ba 3 . The EPR characterizations show similar results for aa 3 at substoichiometric NO/enzyme ratios and for ba 3 , indicating formation of a 6-coordinate heme-NO complex. The presence of a second NO molecule in the aa 3 active site strongly modifies the heme-NO EPR spectrum and can be rationalized by a rotation of the Fe-N-O plane with respect to the histidine that coordinates the heme iron. This proposal is supported by molecular-modeling studies that indicate a ∼63°rotation of heme-bound NO upon binding of a second NO to the close-lying copper center CuB. It is argued that the second NO binds to CuB.

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Section: Resultssupporting

confidence: 91%

“…In particular, binding of CO to this enzyme leads to formation of only 70% of the heme a 3 -CO complex (21,24), with the remaining 30% forming a CuB-CO complex (25). To estimate the extent of NO binding to heme a 3 , the inset of Figure 1B shows the second derivative of the Soret spectra of the unligated and NO-bound forms.…”

Section: Resultsmentioning

confidence: 99%

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa₃ from Paracoccus denitrificans and ba₃ from Thermus thermophilus

et al. 2004

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“…ence on ionic strength, but it should be kept in mind that our experimental approach, in contrast to a turnover assay (19), only follows the initial steps of encounter and ET between the two proteins. Thus the fast kinetics observed here do not depend on a sluggish off-rate and therefore are only moderately affected by ionic strength (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Taking into consideration that the stability of electrostatic interactions is lower at higher temperatures, hydrophobic interactions become more favorable. Kinetic studies have shown that the turnover activity of cytochrome c 552 with ba 3 oxidase becomes faster as ionic strength is decreased (19). On the contrary, P. denitrificans aa 3 cytochrome-c oxidase shows very low turnover rates under low ionic strength conditions, most likely because of the formation of a high affinity electrostatic complex (15).…”

mentioning

confidence: 99%

Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates

Maneg

Ludwig

Malatesta

2003

Journal of Biological Chemistry

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Cytochrome-c oxidase is the terminal enzyme in the respiratory chains of mitochondria and many bacteria and catalyzes the formation of water by reduction of dioxygen. The first step in the cytochrome oxidase reaction is the bimolecular electron transfer from cytochrome c to the homobinuclear mixed-valence Cu A center of subunit II. In Thermus thermophilus a soluble cytochrome c 552 acts as the electron donor to ba 3 cytochrome-c oxidase, an interaction believed to be mainly hydrophobic. In Paracoccus denitrificans, electrostatic interactions appear to play a major role in the electron transfer process from the membrane-spanning cytochrome c 552 . In the present study, soluble fragments of the Cu A domains and their respective cytochrome c electron donors were analyzed by stopped-flow spectroscopy to further characterize the interaction modes. The forward and the reverse electron transfer reactions were studied as a function of ionic strength and temperature, in all cases yielding monoexponential time-dependent reaction profiles in either direction. From the apparent second-order rate constants, equilibrium constants were calculated, with values of 4.8 and of 0.19, for the T. thermophilus and P. denitrificans c 552 and Cu A couples, respectively. Ionic strength strongly affects the electron transfer reaction in P. denitrificans indicating that about five charges on the protein interfaces control the interaction, when analyzed according to the Brøn-sted equation, whereas in the T. thermophilus only 0.5 charges are involved. Overall the results indicate that the soluble Cu A domains are excellent models for the initial electron transfer processes in cytochrome-c oxidases.

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“…They often exhibit significantly different structural features, like for example the cyt c552 from the thermophilic bacterium Thermus thermophilus, which possesses only uncharged residues around the exposed heme edge [25][26][27]. Mainly hydrophobic interactions are thus believed to take place between cyt c552 and the corresponding cyt c oxidases in T. thermophilus [25,28,29]. Bacteria also use diheme proteins to transfer electrons between the respiratory enzyme complexes, such as the cyt c4, which are native electron donors of C family (cbb3) oxidases [30,31].…”

Section: Introductionmentioning

confidence: 99%

Electrochemical study of an electron shuttle diheme protein: The cytochrome c from T. thermophilus

Melin

Schoepp‐Cothenet²,

Abdulkarim³

et al. 2017

Inorganica Chimica Acta

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ABSTRACT:Cytochrome c550, a diheme protein from the thermophilic bacterium Thermus thermophilus, is involved in an alternative respiration pathway allowing the detoxification of sulfite ions. It transfers the two electrons released from the oxidation of sulfite in a sulfite:cytochrome c oxidoreductase (SOR) enzyme to heme/copper oxidases via the monoheme cytochrome c552. It consists of two conformationally independent and structurally different domains (the C-and Nterminal) connected by a flexible linker. Both domains harbor one heme moiety. We report here the redox properties of the full-length protein and the individual C-and N-terminal fragments.We show by UV/Vis and EPR potentiometric titrations that the two fragments exhibit very similar potentials, despite their different environments. In the full-length protein, however, the N-terminal heme is easier to reduce than the C-terminal one, due to cooperative interactions.This finding is consistent with the kinetic measurements which showed that the N-terminal domain only accepts electrons from the SOR. Cytochrome c552 is able to interact with its partners both through electrostatic and hydrophobic interactions as could be shown by measuring efficient electron transfer at gold electrodes modified with charged and hydrophobic groups, respectively. The coupling of electrochemistry with infrared spectroscopy allowed us to monitor the conformational changes induced by electron transfer to each heme separately and to both simultaneously.

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Kinetic Properties of ba₃ Oxidase from Thermus thermophilus: Effect of Temperature

Cited by 76 publications

References 41 publications

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa₃ from Paracoccus denitrificans and ba₃ from Thermus thermophilus

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa₃ from Paracoccus denitrificans and ba₃ from Thermus thermophilus

Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates

Electrochemical study of an electron shuttle diheme protein: The cytochrome c from T. thermophilus

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Kinetic Properties of ba3 Oxidase from Thermus thermophilus: Effect of Temperature

Cited by 76 publications

References 41 publications

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa3 from Paracoccus denitrificans and ba3 from Thermus thermophilus

Different Interaction Modes of Two Cytochrome-c Oxidase Soluble CuA Fragments with Their Substrates

Electrochemical study of an electron shuttle diheme protein: The cytochrome c from T. thermophilus

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Kinetic Properties of ba₃ Oxidase from Thermus thermophilus: Effect of Temperature

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa₃ from Paracoccus denitrificans and ba₃ from Thermus thermophilus

NO Binding and Dynamics in Reduced Heme−Copper Oxidases aa₃ from Paracoccus denitrificans and ba₃ from Thermus thermophilus