Darlene J. Spira

Anion binding to oxidized type 2 depleted and native laccase: A spectroscopically effective model for exogenous ligand binding to the type 3 - type 2 active site

Abstract Chemical and spectroscopic comparison of anion binding to the oxidized type 3 sites in type 2 depleted and native Rhus vernicifera laccase demonstrates that the type 3 - type 2 active site of laccase has an especially high affinity for exogenous ligands. N 3 − , O 2 2− and F− binding and competition, and resonance Raman studies, indicate that this high affinity is not due to strong, equatorial type 2 ligation, but rather to a structurally non-specific role of the type 2 copper in stabilizing anion binding at the type 3 binuclear cupric site. These studies, combined with previous results on half met type 2 depleted laccase, generate a spectroscopically effective model for peroxide birding to the type 3 - type 2 active site.

Preparation and characterization of a stable half met derivative of type 2 depleted Rhus laccase: Exogenous ligand binding to the type 3 site

Abstract We report the preparation and characterization of a stable half met (Cu(II)Cu(I)) type 2 copper depleted derivative of Rhus laccase. Anion binding studies to this mixed valent type 3 protein form indicate no tight binding of anions nor group 1 - group 2 ligand behavior. This suggests that, in contrast to the well-characterized hemocyanins and tyrosinase coupled binuclear sites, exogenous ligands do not appear to bridge the type 3 binuclear copper ions in laccase.

Quantitative Cu(I) determination using X-ray absorption edge spectroscopy: Oxidation of the reduced binuclear copper site in type 2 depleted Rhus laccase

We report a procedure, through difference comparison of X-ray absorption edge spectra, for the quantitative determination of Cu(I) content in copper complexes of mixed oxidation state composition. This technique is tested on copper model systems and then used to quantitatively determine that untreated T2D Rhus laccase contains 70 +/- 15% Cu(I). Whereas excess ferricyanide is demonstrated not to alter the Cu(I) content of the untreated T2D, aqueous peroxide and nitrite at pH 6.0 are shown to oxidize the cuprous type 3 site and generate met T2D protein forms.

On the spectral features associated with peroxide reactivity of the coupled binuclear copper active site in type 2 depleted and native Rhus laccase.

We report herein an X-ray absorption spectroscopic (XAS) determination of the oxidation state of the copper sites in T2D and native Rhus laccase. The increase in intensity of the 330 nm absorption feature which results from peroxide titration of T2D laccase (T3: [Cu(I)Cu(I)], T1: [Cu(II)]) is found to correlate linearly with the percent of oxidation of the binuclear copper site (determined by XAS analysis). This indicates that peroxide oxidizes but does not bind to the T3 site. We have used this correlation to determine that native laccase, as isolated, contains approximately 25% reduced T3 sites and that all spectral changes observed upon peroxide addition to native laccase can be accounted for by oxidation of these reduced sites. The importance of this result to previous reports of peroxide binding at the laccase active site is discussed.

Structural comparison of octahedral MoO22+ complexes of bidentate and linear tetradentate N,S-donor ligands

The structures of MoO2[NH2C(CH3)2CH2S]2 and MoO2[SC(CH3)2CH2NHCH2CH2NHCH2C(CH3)2S] have been determined using X-ray diffraction intensity data collected by counter techniques. MoO2[NH2C(CH3)2CH2S]2 crystallizes in space group Pbca with a = 11.234(3), b = 11.822(3) and c = 20.179(5) A, V = 2680(2) A3 and Z = 8. Its structure is derived from octahedral coordination with cis oxo groups [MoO = 1.705(3) and 1.705(3)], trans thiolate donors cis to the oxo groups [MoS = 2.416(1) and 2.402(1) and N donors trans to oxo [MoN = 2.325(3) and 2.385(4) A]. MoO2[SC(CH3)2CH2NHCH2CH2NHCH2C(CH3)2S] crystallizes in the space group P21/c with a = 10.798(5), b = 6.911(2), c = 20.333(9) A, β = 95.20°, V = 1511(2) A3 and Z = 4. Its structure is very similar to that of MoO2[NH2C(CH3)2CH2S]2 with MoO = 1.714(2) and 1.710(2), MoS = 2.415(1) and 2.404(1) and MoN = 2.316(3) and 2.362(3). The small differences in the geometries of the two compounds are attributed to the constraints of the extra chelate ring in the complex with the tetradentate ligand. The structures in this paper stand in contrast to those reported for complexes of similar ligands wherein steric hindrance produces complexes with a skew trapezoidal bipyramidal structure.

EXAFS investigation of the binuclear cupric site in met T2D Rhus laccase and its azide bound derivative.

EXAFS analysis of met T2D Rhus laccase and its azide bound derivative indicates an average of 0.33 S at 2.09 A and 3-4 N (or O) atoms at 2.00 A per copper atom for the three copper centers. Using the plastocyanin Cu(II) EXAFS spectrum to model the type 1 site in laccase, a difference EXAFS spectrum for the type 3 site is generated; this spectrum enables assignment of the one S ligand in met T2D to the type 1 site and indicates no evidence of a detectable copper scatterer for the coupled binuclear copper site. Implications regarding type 3 optical features and related studies on the hemocyanins are also discussed.

Nitrite reactivity of the binuclear copper site in T2D Rhus laccase: preparation of half met-NO2- T2D laccase and its correlation to half met-NO2- hemocyanin and tyrosinase.

Through chemistry directly comparable to that of the hemocyanins and tyrosinase, half met-NO2- T2D laccase derivatives have been prepared; this NO2- reactivity entails both two electron oxidation of the cuprous binuclear site in deoxy T2D laccase and one electron reduction of the coupled cupric site in the met derivative. However, the labile ligand substitution chemistry and lack of dimer formation in half met-NO2- T2D are in marked contrast to behavior of the simpler binuclear copper containing proteins under analagous conditions. This chemistry supports and extends our earlier studies on the ferrocyanide-generated half met T2D which first indicated an inability of exogenous ligands to bridge the binuclear copper site in laccase.

Selective chemical and physical perturbations for the different copper sites in the multicopper oxidase rhus laccase

Abstract Laccase [1] contains one blue (T1) and one normal (T2) copper which interact with the single binuclear copper (T3) site to couple one-electron oxidations of substrate to the four-electron reduction of dioxygen to water. We here report reversible chemical simplifications and the application of spectroscopic methods particularly suited for the multicopper oxidase which together enable detailed study of the T3 site in laccase and structure-function correlation to the simpler binuclear copper containing proteins, hemocyanin and tyrosinase. Derivatives of the simplified type 2 copper-depleted (T2D) [2] protein form have been prepared which allow systematic variation of the coupled binuclear copper site in the presence of an oxidized T1 center. Spectroscopic study of these derivatives (deoxy, [Cu(I)Cu(I), half met [Cu(I)Cu(II), met [Cu(II)⋯Cu(II), and dimer [Cu(II)Cu(II)]) has allowed determination of the spectral features associated with the binuclear copper centers in laccase. Comparison [3] of deoxy and met T2D identifies the optical features of the binuclear cupric site (ϵ 330 = 2000 M −1 , ϵ 420 = 175 M −1 cm −1 , and ϵ 745 = 150 M −1 cm −1 ); anion binding studies of the EPR-detectable half met derivatives directly probe geometric structure at the cupric T3 center, demonstrating that exogenous ligands are only weakly bound and do not bridge the binuclear coppers [4]. Spectral comparison of the T2D derivatives and their anated forms has demonstrated the existence of allosteric interaction between the T1 and T3 sites. Laser excitation into the deoxy T2D Blue copper CT band results in a 60% reduction of intensity and increased frequency of the ∼380 cm −1 vibration compared to native and met T2D protein, whose spectra are similar. In the EPR, A∥ increase from 37.8 to 42.9 × 10 −4 cm −1 when deoxy is oxidized to met, demonstrating that changes in the T1 copper geometry are linked to oxidation of the T3 copper [3]. Finally, chemical and spectroscopic comparison [5] of anion binding to the oxidized T3 site in met T2D and native laccase indicates a unique T3-T2 intersite interaction which leads to an especially high affinity for exogenous ligands. Ligand competition studies systematically probe the chemical nature of these binding sites and their interdependence, while a combination of variable excitation energy resonance Raman and variable temperature MCD techniques are used to spectroscopically define the role of the T2 copper in greatly increasing ligand binding affinity at the T3 site. L → Cu(II) CT transitions are present for a number of ligated forms of the native enzyme, and resonance Raman enhancement together with mixed isotope studies provide assignment of these features and insight into binding geometry; a comparison to analogous studies of met T2D probes the T2 Cu(II) contribution. Finally, MCD of these features will be presented and their temperature dependence used to distinguish ligand binding to the antiferromagnetically coupled T3 copper centers from binding to the paramagnetic T1 and T2 Cu(II) centers which are predicted to exhibit C terms for which MCD intensity is proportional to 1/T.

X-ray absorption studies of laccase and ceruloplasmin

Abstract Determination of oxidation state is essential to interpretation of the spectroscopy and chemistry of metal ions. We will describe the use of X-ray absorption edges to quantitatively determine the percent of Cu(I) in samples of mixed Cu(I)/Cu(II) composition, and specific applications of this technique to characterization of copper oxidation states in various derivatives of the multicopper oxidases laccase and ceruloplasmin. It has long been recognized that the energy and the shape of an X-ray absorption edge are correlated, respectively, with the effective charge on the absorbing atom and with the geometry of the absorbing site. Cu complexes, in particular, have X-ray absorption edges which change dramatically with metal oxidation state. Cuprous complexes show an intense transition at approximately 8984 eV which is absent for complexes in the +2 oxidation state. In addition, the peak absorbance for Cu(I) complexes is less intense that for Cu(II) complexes. The different edge shapes for Cu(I) and Cu(II) have previously been used as a qualitative indicator of the presence of Cu(I) in a variety of metalloproteins [1–4], and in particular, in a study of the multicopper oxidase Rhus vernicifera laccase [5]. Laccase, which contains four copper ions at its active site, is the least complicated of the multicopper oxidases and is the current focus of our binuclear copper research. While study of the coupled binuclear site in this enzyme is complicated by the presence of blue and normal copper centers, a reversible procedure exists for T2 copper removal to yield type 2 depleted laccase (T2D) [6], wherein the binuclear site is only complicated by the additional blue copper site. Much controversy has ensued over the chemical and spectroscopic properties of the prepared T2D laccase [7]. Our preliminary X-ray absorption edge studies [5] indicated that T2D contained approximately 70% cuprous copper, while H2O2 treated (met) T2D and native laccase contained essentially no Cu(I). Together with EPR evidence that the T1 copper remains oxidized in T2D laccase, these edge studies demonstrated that T2D laccase contains a reduced T3 site which can be reoxidized by peroxide. We will present evidence that the properly normalized difference between two Cu X-ray absorption edges, under certain circumstances, can also be used to quantitatively determine the Cu(I) concentration in a sample [8]. We have studied an extensive series of Cu model complexes in order to determine the limitation of the different-edge technique. We have then used this technique to quantitatively determine the Cu(I) concentration in native and T2D laccase as well as their ferricyanide, nitrite, and hydrogen peroxide treated forms. Preliminary results on ceruloplasmin will also be presented. An Extended X-ray Absorption Fine Structure (EXAFS) study of met and met-N−3 T2D laccase is also discussed. The EXAFS data are analysed to determine the average environment of the three Cu atoms in the T2D laccase derivatives, and also compared with the EXAFS data for plastocyanin, which is an appropriate model for the blue copper site in laccase [9].