Metalloprotein active site structure determination: Synergy between X-ray absorption spectroscopy and X-ray crystallography Julien J.H. Cotelesage a, b , M. Jake Pushie a , Pawel Grochulski b , Ingrid J. Pickering a , Graham N. George a, ⁎ a Molecular and Environmental Sciences Research Group, Department of Geological Sciences, University of Saskatchewan, Saskatoon, SK, Canada S7N 5E2 b Canadian Light Source, 101 Perimeter Road, Saskatoon, SK, Canada S7N 0X4 abstract article info Article history: Received 10 April 2012 Received in revised form 21 June 2012 Accepted 22 June 2012 Available online 6 July 2012 Keywords: X-ray absorption spectroscopy EXAFS Metalloproteins Macromolecular crystallography Structures of metalloprotein active sites derived from X-ray crystallography frequently contain chemical anomalies such as unexpected atomic geometries or elongated bond-lengths. Such anomalies are expected from the known errors inherent in macromolecular crystallography (ca. 0.1–0.2 Å) and from the lack of ap- propriate restraints for metal sites which are often without precedent in the small molecule structure litera- ture. Here we review the potential of X-ray absorption spectroscopy to provide information and perspective which could aid in improving the accuracy of metalloprotein crystal structure solutions. We also review the potential problem areas in analysis of the extended X-ray absorption fine structure (EXAFS) and discuss the use of density functional theory as another possible source of geometrical restraints for crystal structure anal- ysis of metalloprotein active sites. © 2012 Elsevier Inc. All rights reserved. 1. Introduction In recent decades advances in experimental and analysis tech- niques have caused macromolecular X-ray crystallography to mature from a highly specialized method practiced by a few experts to being widely used and the premier structural technique in biochemistry. For large molecules at lower resolution the method is under-determined, so that restraints, and to a lesser extent constraints, are required for solving structures [1]. Thus, interatomic distances in the amino acids are restrained to values taken from small molecule crystal struc- tures [2]. In the case of metal-containing active sites the structures present are often totally unknown, and restraints are thus unavailable [3]. Moreover, because of the limited resolution, and as discussed by Rees and co-workers [4,5], Fourier series termination artifacts can cause problems in determining the positions of light atoms in the vicin- ity of a heavy atom such as a metal ion. Fortunately, other methods can be used to provide supplemental information on the active site struc- tures. The purpose of this paper is to review the contributions that X-ray absorption spectroscopy (XAS) can make to structure determina- tion of metalloprotein active sites. XAS can contribute in three different ways: (i) XAS has greater accuracy of metal to ligand bond-length de- termination; (ii) XAS has a lower tendency for X-ray photo-reduction; and (iii) XAS can be measured on solutions, thereby avoiding artifacts of crystallization. This review will discuss the advantages and limita- tions of XAS relative to crystallography in general, predominantly in the first area. Hans Freeman was well known for his pioneering work on protein crystallography and blue copper proteins [6]. Since the use of XAS in conjunction with crystallography was one of his long-standing interests [7,8], we have therefore selected this topic for our contribution to this volume. 2. Materials and Methods 2.1. X-ray absorption spectroscopy experiments X-ray absorption spectra were collected at the Stanford Synchrotron Radiation Lightsource using the structural molecular biology beamline 7–3, as previously described [9]. 2.2. Density functional theory calculations Density functional theory (DFT) calculations employed the pro- grams Dmol 3 Materials Studio Version 5.5 [10,11]. Geometry optimiza- tion calculations used the Perdew–Burke–Ernzerhof functional [12,13] for both the potential during the self-consistent field procedure, and the energy. Dmol 3 uses numerically derived basis sets [10,11] and these included polarization functions for all atoms. Calculations were spin-unrestricted and all-electron relativistic core potentials were used. Solvation effects were modeled using the Conductor-like Screen- ing Model (COSMO) [14] with a dielectric value representing water (ε =78.39). Convergence was assumed to be achieved when energies differed by less than 2×10 -5 E h , the maximum force was less than 0.004 E h /Å and the maximum displacement was less than 0.005 Å. A maximum step size of 0.3 Å was used. Journal of Inorganic Biochemistry 115 (2012) 127–137 ⁎ Corresponding author at: Molecular and Environmental Sciences Research Group, Department of Geological Sciences, University of Saskatchewan, 114 Science Place, Saskatoon, SK, Canada S7N 5E2. Tel.: +1 306 966 5722, +1 306 966 8593. E-mail address: g.george@usask.ca (G.N. George). 0162-0134/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.jinorgbio.2012.06.019 Contents lists available at SciVerse ScienceDirect Journal of Inorganic Biochemistry journal homepage: www.elsevier.com/locate/jinorgbio