Coordination environment and fluoride binding of type 2 copper in the blue copper oxidase ceruloplasmin.

Dawson, John H.; Dooley, David M.; Gray, Harry B.

doi:10.1073/pnas.75.9.4078

Cited by 24 publications

(7 citation statements)

References 27 publications

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“…The T2 copper exhibits EPR parameters typical for normal copper ( A ∥ = 158−201 × 10 -4 cm -1 ) and no intense features in the visible absorption spectrum. An archetypal feature of the T2 site in the multicopper oxidases is its ability to bind F - with an unusually high affinity, a feature which can easily be identified by the 50 × 10 -4 cm -1 superhyperfine splitting observed in the EPR spectrum ( vide infra ). − The potential of the T2 site has been reported only for R. vernicifera Lc, wherein it is 365 mV . The T3 copper pair has an absorption band at around 330 nm (ε ≈ 5000 M -1 cm -1 ) and is EPR-silent, indicative of a strongly antiferromagnetically coupled Cu(II) pair bridged by a hydroxide.…”

Section: Multicopper Oxidasesmentioning

confidence: 99%

Multicopper Oxidases and Oxygenases

1996

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Section: Multicopper Oxidasesmentioning

confidence: 99%

Multicopper Oxidases and Oxygenases

1996

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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 ).…”

mentioning

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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“…In fungal laccase, plant laccase, ascorbate oxidase, and human Cp it was previously found that two fluoride ions bind selectively to the T2 copper site, one with high affinity and one with low affinity ( − ). This is seen by EPR, in which fluoride-binding induces a superhyperfine splitting of about 50 × 10 -4 cm -1 in the low-field hyperfine line of the T2 copper.…”

Section: Resultsmentioning

confidence: 99%

Investigation of the Anomalous Spectroscopic Features of the Copper Sites in Chicken Ceruloplasmin: Comparison to Human Ceruloplasmin

et al. 1999

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Chicken ceruloplasmin has been previously reported to display a number of key differences relative to human ceruloplasmin: a lower copper content and a lack of a type 2 copper signal by electron paramagnetic resonance (EPR) spectroscopy. We have studied the copper sites of chicken ceruloplasmin in order to probe the origin of these differences, focusing on two forms of the enzyme: "resting" (as isolated by a fast, one-step procedure) and "peroxide-oxidized". From X-ray absorption, EPR, and UV/visible absorption spectroscopies, we have shown that all of the copper sites are oxidized in peroxide-oxidized chicken ceruloplasmin and that none of the type 1 copper sites display the EPR features typical for type 1 copper sites that lack an axial methionine. In the resting form, the type 2 copper center is reduced. Upon oxidation, it does not appear in the EPR spectrum at 77 K, but it can be observed by using magnetic susceptibility, EPR at approximately 8 K, and magnetic circular dichroism spectroscopy. It displays unusually fast relaxation, indicative of coupling with the adjacent type 3 copper pair of the trinuclear copper cluster. From reductive titrations, we have found that the reduction potential of the type 2 center is higher than those of the other copper sites, thus explaining why it is reduced in the resting form. These results provide new insight into the nature of the additional type 1 copper sites and the redox distribution among copper sites in the different ceruloplasmins relative to other multicopper oxidases.

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Coordination environment and fluoride binding of type 2 copper in the blue copper oxidase ceruloplasmin.

Abstract: The electron paramagnetic resonance (EPR) spectra of the blue copper oxidase ceruloplasmin ferroxidase, iron(II

Cited by 24 publications

References 27 publications

Multicopper Oxidases and Oxygenases

Multicopper Oxidases and Oxygenases

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

Investigation of the Anomalous Spectroscopic Features of the Copper Sites in Chicken Ceruloplasmin: Comparison to Human Ceruloplasmin

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