Selective removal of type 2 copper from <i>Rhus vernicifera</i> laccase

Graziani, Maria Teresa; Morpurgo, Laura; Rotilio, Giuseppe; Mondovı̀, Bruno

doi:10.1016/0014-5793(76)80732-6

Cited by 104 publications

(56 citation statements)

References 9 publications

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“…Some samples however show a lower total copper content after EDTA-DMG treatment. It appears therefore that the treatment removes almost selectively the type 2 copper from ascorbate oxidase as reported for lactase [2], though some type 3 copper could be also removed. Since the ment, which causes a 95% decrease of ascorbate oxidase activity.…”

Section: Resultsmentioning

confidence: 56%

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Removal of non‐blue copper from ascorbate oxidase

et al. 1979

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Section: Resultsmentioning

confidence: 56%

“…In order to get new information on this problem, the possibility of a selective removal of the metal with a method described for Rhus vernicifera lactase [2] was investigated. The metal-depleted ascorbate oxidase appears to have lost type 2 copper.…”

Section: Introductionmentioning

confidence: 99%

Removal of non‐blue copper from ascorbate oxidase

et al. 1979

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“…21 We have characterized a type 2 depleted (T2D) derivative of the MCO's where the T2 Cu is reversibly removed and remaining is the T3 (and T1) to react with O 2 . 22 From figure 7B, in contrast to Hc and Tyr, the reduced T3 Cu center does not react with O 2 . 20 As a reference we first consider the reversible binding of O 2 by Hc.…”

Section: O 2 Reactivity: T3 Vs Coupled Binuclear Cu (Hc and Tyr) Sitesmentioning

confidence: 92%

O2 Reduction to H2O by the multicopper oxidases

2008

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In nature the four electron reduction of O 2 to H 2 O is carried out by Cytochrome c Oxidase (CcO) and the multicopper oxidases (MCOs). In the former, Cytochrome c provides electrons for pumping protons to produce a gradient for ATP synthesis, while in the MCOs the function is the oxidation of substrates, either organic or metal ions. In the MCOs the reduction of O 2 is carried out at a trinuclear Cu cluster (TNC). Oxygen intermediates have been trapped which exhibit unique spectroscopic features that reflect novel geometric and electronic structures. These intermediates have both intact and cleaved O-O bonds, allowing the reductive cleavage of the O-O bond to be studied in detail both experimentally and computationally. These studies show that the topology of the TNC provides a unique geometric and electronic structure particularly suited to carry out this key reaction in Nature.The multicopper oxidases (MCOs) couple four 1-electron oxidations of substrate to the four electron reductive cleavage of the O-O bond of dioxygen using a minimum of four Cu atoms (table 1). 1,2 Among these four Cu's is a type 1 (T1) or blue Cu site, characterized by an intense S Cys → Cu(II) charge transfer (CT) transition at around 600 nm in the absorption spectrum and a uniquely small A || in its electron paramagnetic resonance (EPR) spectrum. This is the site of substrate oxidation, and from table 1, the MCOs can be divided into two classes depending upon the identity of the substrate. For enzymes such as laccase 3 and ascorbate oxidase, 4 redox active organic molecules which can interact weakly with the enzyme provide the electrons. For MCOs like Fet3p 5 and Ceruloplasmin, 6 the substrate is a metal ion (ferrous in these cases) which binds tightly to a substrate binding site. As shown in figure 1, these substrate binding sites are located near the His ligands of the T1 Cu center. The electron from substrate is first transferred to the T1 and then over >13Å through a Cys-His pathway to a trinuclear Cu cluster (TNC) where O 2 is reduced to water (vide infra). 7 We first consider the electron transfer (ET) pathways to the TNC. ET PathwaysHere we focus on the Fe(II) binding site of the enzyme Fet3p, which is involved in the uptake of iron by yeast. 8 (Studies on this enzyme were performed in collaboration with Prof. Dan Kosman and coworkers.) A variable-temperature, variable-field magnetic circular dichroism (VTVH MCD) methodology we developed in other studies was applied to probe this ferrous site. [9][10][11] From figure 2A dark blue, there is a characteristic feature at 8900 cm −1 in the MCD spectrum corresponding to Fe(II) binding with a high affinity (K B > 10 5 M −1 , from MCD titration studies) to a 6 coordinate site in the protein. 12 In the light blue spectrum this feature is eliminated and a peak at 9700 cm −1 , corresponding to aqueous Fe(II) (green) is observed when Zn(II) is first bound to the substrate site, inhibiting ferroxidase activity.From mutagenesis studies we have found that three carboxylates are involved...

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“…Japanese Rhus vernicifera Lc and its T2 depleted derivative were purified and assayed according to published procedures (35,36). Rapid freeze-quenched (RFQ) NI samples were obtained as reported in ref.…”

Section: Methodsmentioning

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.

106

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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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Selective removal of type 2 copper from Rhus vernicifera laccase

Cited by 104 publications

References 9 publications

Removal of non‐blue copper from ascorbate oxidase

Removal of non‐blue copper from ascorbate oxidase

O2 Reduction to H2O by the multicopper oxidases

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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