Spectroscopic and Electronic Structure Studies of the Trinuclear Cu Cluster Active Site of the Multicopper Oxidase Laccase:  Nature of Its Coordination Unsaturation

Quintanar, Liliana; Yoon, Jungjoo; Aznar, Constantino P.; Palmer, Amy E.; Andersson, K. Kristoffer; Britt, R. David; Solomon, Edward I.

doi:10.1021/ja0421405

Cited by 123 publications

(169 citation statements)

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“…The inhibition mechanism of Lac was investigated by using spectrophotometrically method and a combination of spectroscopic and DFT studies. [17][18][19][20] Voltammetric studies on the halide inhibition was carried out, which suggested the ability to differentiate the inhibition modes of Lac by halides. 5,13 However, the mechanism of inhibition has not yet been clarified.…”

Section: Resultsmentioning

confidence: 99%

“…16 Lac electrodes in biosensors can be inhibited by Cl ¹ , which affects their performance under physiological conditions. [4][5][6]12,13,[17][18][19][20] Intracellular fluid contains little Cl ¹ (³3 © 10 ¹3 mol dm…”

Section: ¹1mentioning

confidence: 99%

“…The value of K was ³10 times smaller than that reported in optical adsorption and electron paramagnetic resonance spectral studies in pH 5.5 buffer solution. 18,19 The halide ion inhibition behaviors of the SWCNT and carbon paper electrodes can be explained by the differing Cl ¹ and F ¹ inhibition mechanisms. Very fast heterogeneous electron transfer of Lac occurred at the SWCNT interface.…”

Section: ¹3mentioning

confidence: 99%

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Bioelectrocatalytic Oxygen Reaction and Chloride Inhibition Resistance of Laccase Immobilized on Single-walled Carbon Nanotube and Carbon Paper Electrodes

2016

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The rate of heterogeneous direct electron transfer of laccase immobilized on single-walled carbon nanotube (SWCNT) and carbon paper electrodes was evaluated by cyclic voltammetry and background-current-corrected steady-state linear voltammetry. These rates indicated that the molecular orientation of laccase immobilized on the SWCNT electrode was more favorable for direct electron transfer, than that of laccase immobilized on the carbon paper electrode. The inhibition of the bioelectrocatalytic O 2 reduction current of the two electrodes by chloride and fluoride were tested. The results indicated differing inhibition mechanisms by these two halides. Laccase immobilized on the SWCNT electrode exhibited high stability and high resistance to chloride inhibition.

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Section: Resultsmentioning

confidence: 99%

Section: ¹1mentioning

confidence: 99%

Section: ¹3mentioning

confidence: 99%

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Bioelectrocatalytic Oxygen Reaction and Chloride Inhibition Resistance of Laccase Immobilized on Single-walled Carbon Nanotube and Carbon Paper Electrodes

2016

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“…This has allowed us, for the first time, to monitor time-resolved MCD resulting from in situ chemical manipulation of a metalloprotein sample. Furthermore, we report the parallel development of an electrochemical cell using a three-electrode configuration with physically separated working and counter electrodes, allowing true potentiometric titration to be performed within the bore of the MCD solenoid.Ó 2011 Elsevier Inc. All rights reserved.Magnetic circular dichroism (MCD) 1 spectroscopy, at ultraviolet-visible and near-infrared wavelengths (185-2000 nm), has proved to be invaluable in the study of metalloproteins containing cofactors such as heme [1][2][3], non-heme iron [4], iron-sulfur clusters [5][6][7][8], cobalt [9,10], nickel [11][12][13], and copper [14,15]. The technique measures the apparent circular dichroism (CD) induced by a magnetic field [16].…”

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Probing a Complex of Cytochromecand Cardiolipin by Magnetic Circular Dichroism Spectroscopy: Implications for the Initial Events in Apoptosis

et al. 2011

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a b s t r a c tMagnetic circular dichroism (MCD) spectra, at ultraviolet-visible or near-infrared wavelengths (185-2000 nm), contain the same transitions observed in conventional absorbance spectroscopy, but their bisignate nature and more stringent selection rules provide greatly enhanced resolution. Thus, they have proved to be invaluable in the study of many transition metal-containing proteins. For mainly technical reasons, MCD has been limited almost exclusively to the measurement of static samples. But the ability to employ the resolving power of MCD to follow changes at transition metal sites would be a potentially significant advance. We describe here the development of a cuvette holder that allows reagent injection and sample mixing within the 50-mm-diameter ambient temperature bore of an energized superconducting solenoid. This has allowed us, for the first time, to monitor time-resolved MCD resulting from in situ chemical manipulation of a metalloprotein sample. Furthermore, we report the parallel development of an electrochemical cell using a three-electrode configuration with physically separated working and counter electrodes, allowing true potentiometric titration to be performed within the bore of the MCD solenoid.Ó 2011 Elsevier Inc. All rights reserved.Magnetic circular dichroism (MCD) 1 spectroscopy, at ultraviolet-visible and near-infrared wavelengths (185-2000 nm), has proved to be invaluable in the study of metalloproteins containing cofactors such as heme [1][2][3], non-heme iron [4], iron-sulfur clusters [5][6][7][8], cobalt [9,10], nickel [11][12][13], and copper [14,15]. The technique measures the apparent circular dichroism (CD) induced by a magnetic field [16]. Despite similarities in the instrumentation used, the observation of MCD is not dependent on the chirality of the protein; the method is equally applicable to racemic model complexes [17]. The magnetic field will always induce signals across wavelengths at which the substance absorbs. Thus, MCD spectra contain the same electronic transitions observed in conventional absorbance spectroscopy, but the bisignate nature of the spectrum provides enhanced resolution and greater detail. This spectral detail offers an unmatched fingerprinting capability that can, for example, identify the spin and oxidation states of heme groups [1] and distinguish among the variety of iron-sulfur centers found in biological molecules [18][19][20].Metalloprotein MCD can be measured at ambient or cryogenic temperatures. The latter requires adulteration with glassing agents [16] but has generally been preferred because MCD intensity from paramagnetic centers increases dramatically at low temperature. Thus, most metalloprotein MCD reported has been measured in glasses at temperatures of approximately 4.2 K. Hemoproteins represent the significant exception, giving rise to appreciable MCD at high temperatures [1]. Thus, ambient temperature MCD is used to diagnose spin state, oxidation state, and (in the case of low-spin Fe(III) hemes) axial ligation [21].As ...

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“…Alternatively, F Ϫ -bound forms of different MCOs exhibit very similar spectral features, where F Ϫ binds strongly (K b Ϸ 10 5 M Ϫ1 ) at the 3 -position of the trinuclear site (13,20,28,30). With its small size and high affinity, F Ϫ would bind to the trinuclear site unobstructed by the protein environment (N 3 Ϫ has an Ϸ200-fold lower K b ).…”

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confidence: 99%

The two oxidized forms of the trinuclear Cu cluster in the multicopper oxidases and mechanism for the decay of the native intermediate

Yoon

Liboiron

Sarangi

et al. 2007

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

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Multicopper oxidases (MCOs) catalyze the 4e ؊ reduction of O2 to H 2O. The reaction of the fully reduced enzyme with O2 generates the native intermediate (NI), which undergoes a slow decay to the resting enzyme in the absence of substrate. NI is a fully oxidized form, but its spectral features are very different from those of the resting form (also fully oxidized), because the type 2 and the coupled-binuclear type 3 Cu centers in the O 2-reducing trinuclear Cu cluster site are isolated in the resting enzyme, whereas these are all bridged by a 3-oxo ligand in NI. Notably, the one azide-bound NI (NIAz) exhibits spectral features very similar to those of NI, in which the 3-oxo ligand in NI has been replaced by a 3-bridged azide. Comparison of the spectral features of NI and NI Az, combined with density functional theory (DFT) calculations, allows refinement of the NI structure. The decay of NI to the resting enzyme proceeds via successive proton-assisted steps, whereas the ratelimiting step involves structural rearrangement of the 3-oxo-bridge from inside to outside the cluster. This phenomenon is consistent with the slow rate of NI decay that uncouples the resting enzyme from the catalytic cycle, leaving NI as the catalytically relevant fully oxidized form of the MCO active site. The all-bridged structure of NI would facilitate electron transfer to all three Cu centers of the trinuclear cluster for rapid proton-coupled reduction of NI to the fully reduced form for catalytic turnover.laccase ͉ superexchange ͉ electron paramagnetic resonance ͉ magnetic circular dicroism ͉ density functional theory M ulticopper oxidases (MCOs) catalyze the 4e Ϫ reduction of O 2 to H 2 O by using four Cu centers. The electrons are taken up at the blue type 1 (T1) Cu site and transferred Ϸ13 Å to the trinuclear Cu cluster, composed of a normal type 2 (T2) and a coupled-binuclear type 3 (T3) site, where the O 2 reduction occurs (1, 2). The T2 Cu site is held in the protein by two His and has a water-derived OH Ϫ ligand external to the cluster, whereas the OH Ϫ -bridged T3 Cu site is held by three His on each Cu. The reaction of O 2 with the fully reduced enzyme generates the native intermediate (NI) (3-7). A combination of Cu K-edge x-ray spectroscopy (XAS) and magnetic circular dichroism (MCD) has unambiguously demonstrated that NI is a fully oxidized form with fully reduced O 2 (3). Recent model studies combined with calculations have further demonstrated that the three Cu(II) centers in the trinuclear site are all bridged by a 3 -oxo ligand (8, 9). In the absence of reducing substrate, NI slowly decays to the resting enzyme, in which the one remaining O atom of the O 2 is terminally bound as OH Ϫ to the T2 site, as indicated by 18 O isotope ratio MS (IRMS) (10, 11) and 17 O EPR (12) experiments. Thus, this process requires the 3 -oxo-bridged NI to undergo a large rearrangement. Importantly, the slow rate of NI decay conflicts with the much higher turnover number, indicating that the resting enzyme is not involved in the catalytic cyc...

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Spectroscopic and Electronic Structure Studies of the Trinuclear Cu Cluster Active Site of the Multicopper Oxidase Laccase: Nature of Its Coordination Unsaturation

Cited by 123 publications

References 66 publications

Bioelectrocatalytic Oxygen Reaction and Chloride Inhibition Resistance of Laccase Immobilized on Single-walled Carbon Nanotube and Carbon Paper Electrodes

Bioelectrocatalytic Oxygen Reaction and Chloride Inhibition Resistance of Laccase Immobilized on Single-walled Carbon Nanotube and Carbon Paper Electrodes

Probing a Complex of Cytochromecand Cardiolipin by Magnetic Circular Dichroism Spectroscopy: Implications for the Initial Events in Apoptosis

The two oxidized forms of the trinuclear Cu cluster in the multicopper oxidases and mechanism for the decay of the native intermediate

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