James E. Hahn

Observation of an electric quadrupole transition in the X-ray absorption spectrum of a Cu(II) complex

Abstract The polarized X-ray absorption cross section of the ls → 3d transition in a square planar CuCl2−4 complex has been measured with respect to rotation about an axis normal to the CuCl4. The cross section exhibits four-fold periodicity about this axis, indicating that the transition is primarily due to coupling with the electric quadrupole component of the radiation. The vibronically allowed dipole transition is approximately one-third as the quadrupolar cross section. These observations are in agreement with SCF Xα multiple-scattered wave calculations, and may have implications for the intepretation of other X-ray absorption spectra. The half-filled d orbital is shown to have the angular characteristics of dx2−y2.

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.

X-ray absorption spectroscopy of 3-Fe clusters in FeS proteins

Abstract Fe EXAFS and edge spectroscopy have been used to characterize the 3-Fe clusters in aconitase and Azotobacter vinelandii ferredoxin I (Av Fd I). Fe EXAFS of a frozen solution of oxidized (unactivated) beef heart aconitase indicates a ‘compact’ cluster structure with FeFe distances of ca. 2.7 A.In combination with independent iron and acid-labile sulfur determinations, these results allow us to propose a structure for the aconitase 3-Fe cluster with a stoichiometry of [3Fe-4S]. Single crystal polarized X-ray absorption spectroscopy was performed on Av Fd I crystals with the goal of distinguishing FeFe scattering contributions from the 4-and 3-Fe clusters. Crystallographic results on this protein suggest and ‘extended’ structure for the 3-Fe cluster with ca. 4.1 A Fefe distances. Polarized spectra were recorded with the X-ray polarization vector both normal to and parallel to the average plane of the irons in the 3-Fe clusters. The Fourier transforms were compared and found to be identical in the region in which a 4.1 A scattering peak would be expected. Thus, the long (ca. 4.1 A FeFe distances cannot be detected in Av Fd I crystals at room temperature. Low-temperature solution work is planned to look for evidence of this interaction.

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].