Low-temperature magnetic circular dichroism studies of native laccase: spectroscopic evidence for exogenous ligand bridging at a trinuclear copper active site.

Allendorf, Mark D.; Spira, Darlene J.; Solomon, Edward I.

doi:10.1073/pnas.82.10.3063

Cited by 118 publications

(88 citation statements)

References 24 publications

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“…Structural descriptions of R Az have been available from both spectroscopy and crystallography. Spectroscopy first determined that the azide must bridge the T2 and T3 Cu centers in R Az (14,15). This in fact provided the first evidence for the presence of the trinuclear Cu clusters in biology, which was confirmed by the first crystal structure of an MCO, ascorbate oxidase (AO) (16).…”

mentioning

confidence: 78%

“…4, band 6) and R Az reflect the similar T2 site geometries in the two forms. Alternatively, the N 3 Ϫ nb 3 Cu T3␣/T3␤ CT transitions at Ϸ29,400 cm Ϫ1 in the absorption and CD spectra of R Az (14) are Ϸ3,000-6,000 cm Ϫ1 higher in energy than those of NI Az , consistent with having the additional coordination of the OH Ϫ bridging ligand to the T3 Cu atoms in R Az (27).…”

mentioning

confidence: 82%

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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one azide-bound NI (NIAz) exhibits spectral features very similar to those of NI, in which the 3-oxo ligand in NI has been replaced by a 3-bridged azide.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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mentioning

confidence: 78%

mentioning

confidence: 82%

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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“…It is well known that the optical absorption spectra of metalloproteins depend markedly on temperature (Knowles et al 1975;San Biagio et al 1977;Dooley et al 1979;Solomon et al 1980;Browett and Stillman 1984;Allendorf et al 1985;Cordone et al 1986;Leone et al 1987;Di Iorio et al 1991;Vitrano et al 1993); as an example we shown in Fig. 1 the Sorer band of Asian elephant MbCO at various temperatures in the range 300-20 K. Three main effects are observed on lowering the temperature: i) halfwidth narrowing, ii) peak frequency shift, iii) integrated intensity variations.…”

Section: Introductionmentioning

confidence: 84%

Low temperature optical absorption spectroscopy: an approach to the study of stereodynamic properties of hemeproteins

et al. 1995

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In this short review we show how suitable analysis of the temperature dependence of the optical absorption spectra of metalloproteins can give insight into their stereodynamic properties in the region of the chromophore. To this end, the theory of coupling between an intense allowed electronic transition of a chromophore and Franck-Condon active vibrations of the nearby atoms is applied to the Soret band of hemeproteins to obtain an analytical expression suitable for fitting the spectral profile at various temperatures. The reported approach enables one to separate the various contributions to the overall bandwidth together with the parameters that characterize the vibrational coupling. The thermal behavior of these quantities gives information on the dynamic properties of the active site and on their dependence upon protein structure and ligation state. The Soret band of hemeproteins appears to be coupled to high frequency vibrational modes of the heme group (as already shown by resonance Raman spectroscopy) and to a "bath" of low frequency modes most likely deriving from the bulk of the protein. For the deoxy derivatives inhomogeneous broadening arising from conformational heterogeneity appears to contribute substantially to the linewidth. The data indicate the onset, at temperatures near 180 K, of large scale anharmonic motions that can be attributed to jumping among different conformational substates of the protein.

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“…There is experimental evidence in earlier studies that the type 2 copper is close to the type 3 copper and forms a trinuclear active copper site [81][82][83][84][85][86]. Solomon and associates described this metal binding site as a trinuclear active site, based on spectroscopic studies of azide binding to tree laccase [85,86]. In ascorbate oxidase, the putative binding site for the reducing substrate is the type 1 copper [43].…”

Section: Native Oxidized Enzymementioning

confidence: 98%

“…The binding of azide in laccase as well as to ascorbate oxidase has been studied extensively by Solomon and co-workers [101,[105][106][107] and by Marchesini and associates [108,109] by spectroscopic techniques. The derived spectroscopic models involve the binding of two azide molecules for laccase and three azide molecules for ascorbate oxidase with different affinities.…”

Section: Azide Form Of Ascorbote Oxidosementioning

confidence: 99%

Metal sites in small blue copper proteins, blue copper oxidases and vanadium-containing enzymes

Messerschmidt

1998

Structure and Bonding

102

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The coordination geometries of metal sites in cupredoxins, mutants and metal derivatives of cupredoxins, multi-copper oxidases and a vanadium-containing chloroperoxidase as derived from X-ray crystallography are described. Correlations with their spectroscopic, electrochemical, electron transfer and catalytic properties are discussed. X-ray crystallography, EPR and Resonance Raman spectroscopy of copper sites in cupredoxins and mutants have led to a classification ranging from type 1 trigonal, type 1 distorted tetrahedral, type 1.5 to type 2. The mutation of copper ligands in azurin or amino acids close to the copper site changes the redox potential in a range of_+140 mV, only. The high redox potential of rusticyanin of 680 mV (azurin, 380 mV) should be mainly due to the special protein environment of the copper site (high proportion of hydrophobic residues).The type 1 and trinuclear copper centres of the multi-copper oxidases ascorbate oxidase, laccase and ceruloplasmin are presented. The metal sites of type 2 depleted, fully-reduced, peroxide and azide forms of ascorbate oxidase, as determined by X-ray crystallography, are discussed in terms of the mechanistic properties of these enzymes.The first X-ray structure of a vanadium-containing protein, namely of a chloroperoxidase from the fungus Curvularia inaequalis, is briefly discussed. The protein fold is mainly a-helical with two four-helix bundles. In the X-ray structure, which is an azide:enzyme complex, the vanadium exhibits a simple unexpected coordination geometry, namely, a trigonal bipyramidal coordination with three non-protein oxygen ligands (VO3 group), one nitrogen ligand from a histidine and one nitrogen from the exogenous azide ligand.Keywords: protein crystallography, small blue copper proteins, multi-copper oxidases, vanadium-containing enzyme, electron transfer 1 plastocyanin from Populus nigra [33,34] plastocyanin from Enteromorpha prolifera [35] plastocyanin from Populus nigra, Hg substituted [36] pseudoazurin from Alcaligenesfaecalis [37,38] amicyanin from Paracoccus denitrificans [39] amicyanin from Thiobacillus versutus [40] cucumber basic protein [41] cucumber stellacyanin [6] azurin from AD containing cadmium [42] copper-containing nitrite reductase from Achromobacter cycloclastes [15] ascorbate oxidase from Cucurbita pepo medullosa [43] mercury substituted type 1 site

show abstract

Low-temperature magnetic circular dichroism studies of native laccase: spectroscopic evidence for exogenous ligand bridging at a trinuclear copper active site.

Cited by 118 publications

References 24 publications

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

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

Low temperature optical absorption spectroscopy: an approach to the study of stereodynamic properties of hemeproteins

Metal sites in small blue copper proteins, blue copper oxidases and vanadium-containing enzymes

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