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

Maneg, Oliver; Ludwig, Bernd; Malatesta, Francesco

doi:10.1074/jbc.m307594200

Cited by 42 publications

(63 citation statements)

References 38 publications

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“…The soluble fragment herein studied comprises only the soluble cupredoxin domain of subunit II, where the transmembrane helix is not part of the purified protein. This fragment retains the structure observed in the whole oxidase (32,45) and it is competent for ET with its biological redox partner (46,47). Given that residues 1-42 are absent in our protein construct, residue numbering starts at 43 in the results presented here, following that employed in the X-ray structure of oxidized holoCu A [Protein Data Bank (PDB) ID 2CUA] (32).…”

Section: Resultsmentioning

confidence: 99%

Flexibility of the metal-binding region in apo-cupredoxins

Zaballa

Abriata

Donaire

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Protein-mediated electron transfer is an essential event in many biochemical processes. Efficient electron transfer requires the reorganization energy of the redox event to be minimized, which is ensured by the presence of rigid donor and acceptor sites. Electron transfer copper sites are present in the ubiquitous cupredoxin fold, able to bind one or two copper ions. The low reorganization energy in these metal centers has been accounted for by assuming that the protein scaffold creates an entatic/rack-induced state, which gives rise to a rigid environment by means of a preformed metal chelating site. However, this notion is incompatible with the need for an exposed metal-binding site and protein-protein interactions enabling metallochaperone-mediated assembly of the copper site. Here we report an NMR study that reveals a high degree of structural heterogeneity in the metal-binding region of the nonmetallated Cu A -binding cupredoxin domain, arising from microsecond to second dynamics that are quenched upon metal binding. We also report similar dynamic features in apo-azurin, a paradigmatic blue copper protein, suggesting a general behavior. These findings reveal that the entatic/rack-induced state, governing the features of the metal center in the copper-loaded protein, does not require a preformed metal-binding site. Instead, metal binding is a major contributor to the rigidity of electron transfer copper centers. These results reconcile the seemingly contradictory requirements of a rigid, occluded center for electron transfer, and an accessible, dynamic site required for in vivo copper uptake.metalloproteins | nuclear magnetic resonance | protein dynamics

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Section: Resultsmentioning

confidence: 99%

Flexibility of the metal-binding region in apo-cupredoxins

Zaballa

Abriata

Donaire

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The binuclear CuA center of N 2 OR is located in a cupredoxin-like domain similar to that found in cytochrome c oxidase and which in both cases constitutes the proposed docking and electron entry site from the small electron donor and enables transfer to the catalytic center [11,16,17]. The CuZ center is located in the N-terminal domain, which adopts a seven-blade b-propeller fold.…”

Section: Introductionmentioning

confidence: 99%

The electron transfer complex between nitrous oxide reductase and its electron donors

Dell’Acqua

Moura

et al. 2011

J Biol Inorg Chem

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Identifying redox partners and the interaction surfaces is crucial for fully understanding electron flow in a respiratory chain. In this study, we focused on the interaction of nitrous oxide reductase (N 2 OR), which catalyzes the final step in bacterial denitrification, with its physiological electron donor, either a c-type cytochrome or a type 1 copper protein. The comparison between the interaction of N 2 OR from three different microorganisms, Pseudomonas nautica, Paracoccus denitrificans, and Achromobacter cycloclastes, with their physiological electron donors was performed through the analysis of the primary sequence alignment, electrostatic surface, and molecular docking simulations, using the bimolecular complex generation with global evaluation and ranking algorithm. The docking results were analyzed taking into account the experimental data, since the interaction is suggested to have either a hydrophobic nature, in the case of P. nautica N 2 OR, or an electrostatic nature, in the case of P. denitrificans N 2 OR and A. cycloclastes N 2 OR. A set of well-conserved residues on the N 2 OR surface were identified as being part of the electron transfer pathway from the redox partner to N 2 OR (Ala495, Asp519, Val524, His566 and Leu568 numbered according to the P. nautica N 2 OR sequence). Moreover, we built a model for Wolinella succinogenes N 2 OR, an enzyme that has an additional c-type-heme-containing domain. The structures of the N 2 OR domain and the c-type-heme-containing domain were modeled and the full-length structure was obtained by molecular docking simulation of these two domains. The orientation of the c-type-heme-containing domain relative to the N 2 OR domain is similar to that found in the other electron transfer complexes.

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“…Cytochrome c 552 and Azu DNA (10¯M each) were mixed under an argon atmosphere. 19 The time course of reduced cytochrome c 552 was traced at the Q-band (552 nm) to determine the apparent ET rate constants. Azu DNA in the CO CooA bound form was prepared by mixing the equivalent amount of 24¯M CO CooA and 20¯M Azu DNA in a CO-saturated solution immediately prior to starting the ET reaction.…”

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confidence: 99%

“…The reaction was monitored by a time course at the Q-band of cytochrome c 552 , corresponding to the oxidation from iron(II) to iron(III) states. 19 Differential spectra upon the ET reaction from iron(II) cytochrome c 552 to Azu DNA of the Cu(II) state in the presence of CO-unbound CooA are shown in Figure 3. Disappearance of bands at max = 423, 528, and 552 nm with an isosbestic point at 412 nm indicates the exclusive oxidation of iron(II) cytochrome c 552 to the iron(III) state under the present reaction conditions.…”

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confidence: 99%

Azurin–DNA Conjugate with the Binding Motif of a Transcriptional Regulator, CooA: CO-dependent Modulation of the Electron-transfer Reaction

et al. 2014

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An azurin DNA conjugate (Azu DNA) where the DNA part contains the binding sequence of a carbon monoxide (CO)-dependent transcriptional regulator, CooA, has been prepared. CooA in the CO-bound form (CO CooA) showed specific binding to Azu DNA. The association of CO CooA with Azu DNA affects the electron transfer (ET) between Azu DNA and its redox partner protein, cytochrome c 552 . Consequently, the CO-dependent modulation of ET was achieved. This could be a potential platform for a novel biobased sensor, which exploits the transcriptional regulator as a detection module and whose readout is immediately transduced to a readily processable electronic signal.A transcriptional regulator is a member of signal transducer proteins that specifically sense an environmental stress to treat it by regulating the activity of the appropriate proteins at the transcriptional level. In biological systems, various transcriptional regulators have evolved to respond to all kinds of stresses, which include not only chemical substances but also physical stimuli such as light, 1,2 heat, 3 and osmotic pressure. 4,5 In general, stress detection by transcriptional regulators is highly sensitive and specific, which are essential in eliminating stresses and retaining homeostasis in living cells. Meanwhile, these properties appear attractive in light of constructing a novel biobased sensor device. A whole cell bacterial sensor is a well-known example that has succeeded in correlating the sensibility of the transcriptional regulator with the activity of a reporter protein. 6 9 In the whole cell sensor, the gene of the reporter protein is fused with the promoter system of the transcriptional regulator so as to reflect the stress detection on the expression level of the reporter protein. Fluorescence and colorimetric reaction by the expressed reporter protein are major choices for the final quantification of the stress level. The bacterial biosensor appears simple and versatile because of the in situ use of the transcriptional regulators in living cells. The progress in this field is, however, sluggish till date due to the difficulty in controlling unexpected responses of the biosensor cell to a target stress under incomplete biological information regarding inherent repression mechanisms of stress. 9 Construction of a similar sensing system in vitro is a potential solution to avoid the problem associated with bacterial biosensors. This, however, requires replacement of the reporter protein system exploiting the physiological function with a facile molecular-base signal transducer in vitro.We have recently developed a signal transduction mechanism consisting of a pair of electron-transfer proteins (azurin 10 and cytochrome c 552 11 from Alcaligenes xylosoxidans and Thermus thermophilus, respectively) and a heat-responsive polymer, poly(N-isopropylacrylamide) (PNIPAM), 12 which is introduced on the hydrophobic surface of azurin to modulate protein protein interaction, prerequisite of the electron-transfer (ET) step. 13 Consequently, the appar...

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

Cited by 42 publications

References 38 publications

Flexibility of the metal-binding region in apo-cupredoxins

Flexibility of the metal-binding region in apo-cupredoxins

The electron transfer complex between nitrous oxide reductase and its electron donors

Azurin–DNA Conjugate with the Binding Motif of a Transcriptional Regulator, CooA: CO-dependent Modulation of the Electron-transfer Reaction

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