X-ray Magnetic Circular Dichroism Sum Rule Analysis of the Blue Copper Site in Plastocyanin. A Probe of Orbital and Spin Angular Momentum Hongxin Wang, ²,‡ C. Bryant, ²,‡ D. W. Randall, § L. B. LaCroix, § E. I. Solomon, § M. LeGros, ‡ and S. P. Cramer* ,²,‡ Department of Applied Science, UniVersity of California, DaVis, California 95616, Physical Biosciences DiVision, Lawrence Berkeley National Laboratory, Berkeley, California 94720, and Department of Chemistry, Stanford UniVersity, Stanford, California 94305 ReceiVed: April 30, 1998; In Final Form: July 21, 1998 Cu L edge X-ray magnetic circular dichroism (XMCD) spectroscopy has been used to study the electronic structure of the blue copper site in plastocyanin. Application of the orbital angular momentum sum rule leads to an experimental (Cu 3d specific) orbital moment 〈L z 〉 of 0.07 ( 0.02 p/Cu atom. This compares to 0.054 p derived from the EPR measurements and 0.059 p/Cu atom from a SCF-XR-SW calculation. Application of the spin angular momentum sum rule leads to an experimental spin moment 〈S z 〉 of 0.18 ( 0.02 p/Cu atom, compared to 0.21 p/Cu atom by SCF-XR-SW. XMCD sum rule analysis should be a useful probe of electronic structure for many other problems in inorganic and bioinorganic chemistry. Introduction X-ray magnetic circular dichroism (XMCD) has recently emerged as a powerful element and site-specific probe of electronic and magnetic structure. 1,2 The development of ligand field multiplet calculations 3 and of X-ray “sum rules” 4-6 allows detailed predictions to be made about orbital and spin angular momentum for particular elements and oxidation states in complex samples. Although XMCD applications are now common in materials science and especially magnetic technol- ogy, 7 its use in biological and inorganic chemistry 8 has been hindered by technical problems associated with the spectroscopy of dilute and paramagnetic metal centers. In this paper we present XMCD spectra for the blue Cu site in plastocyanin, 9,10 recorded with a new dilution refrigerator system and 30-element fluorescence detector. 11,12 Plastocyanin is an ideal system for testing fluorescence-detected XMCD sum rule analysis because its electronic structure is well understood from a variety of experimental and theoretical approaches. 9,10 The half-occupied HOMO is highly covalent with only ∼42% Cu d x 2 -y 2 character. 9 Our XMCD results are compared with EPR data and electronic structure calculations, and a variety of potential applications are discussed. Experimental Section The sample, plastocyanin, was isolated from spinach by previously published methods 13 to a final concentration of 0.8 mM in pH 7 potassium phosphate buffer. A drop (20-50 μL) of the sample was placed on a gold-plated copper sample holder and allowed to dry over a period of 2 h at room temperature in air. During the experiment, the time of beam-on was controlled carefully to minimize the photoreduction of Cu(II) to Cu(I). In the last spectra taken, we estimate less than 10% Cu(I) presence in plastocyanin by monitoring the changes in the minor L edge feature (at about 938 eV). On the other hand, Cu(I) has no XMCD effect. The XMCD experiments were performed at the SSRL bend magnet beamline 8-2 using the 1100 line/mm grating. Ellipti- cally polarized X-rays were obtained by moving the first mirror above or below the electron orbit. 14 On the basis of previous calibration experiments on polarized ferromagnetic samples, the optimum XMCD signal was obtained at a beam takeoff angle corresponding to a circular polarization of 85 ( 5%. The energy resolution is estimated to be 1 eV at the Cu L edge with these elliptically polarized X-ray beams (with both slits at 50 μm). The XMCD apparatus uses a 76 cm split-coil 2 T supercon- ducting magnet surrounded by a UHV chamber maintained at ∼5 × 10 -9 Torr. A 30-element windowless Ge fluorescence detector 11,15 is inserted horizontally between the two coils, perpendicular to the photon beam path. The coldfinger of a He 3 /He 4 dilution refrigerator enters the magnet bore from the top of the chamber, and the sample is attached to the coldfinger at the center of the magnet bore. Samples were introduced into the magnet bore through a vacuum load lock and screwed into the coldfinger using a removable sample insertion device. The lowest operating temperature observed in our experiments was on the order of 400 mK, as measured by a carbon resistance thermometer and by a magnetization curve. Individual scans were taken over the Cu L edges using 0.2 eV steps at 6 s per point integration time. One set of 20 scans was taken with right circular polarization. Every two scans, the magnetic field was switched between -2 and +2 T. A second set of 20 scans was then taken with left circular polarization, again alternating the sign of the magnetic field. The apparent XMCD effect did reverse with opposite beam polarization. For measurement of the magnetization curves, a similar procedure was followed, collecting only the L 3 edge region, using between 0.1 and 2.0 T. The raw data (fluorescence signal, F) were divided by an internal I 0 monitored by the intensity of oxygen K fluorescence (proportional to the beam flux). * To whom correspondence should be addressed. ² University of California. ‡ Lawrence Berkeley National Laboratory. § Stanford University. 8347 J. Phys. Chem. B 1998, 102, 8347-8349 S1089-5647(98)02106-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 10/15/1998