The association of copper to Riboflavin Binding Protein (RBP) from egg white has been studied by electron paramagnetic resonance (EPR) and X-ray absorption (XAS) spectroscopies. The type II site contains a mix of copper I and II in an oxygen rich environment

Bencze, Krisztina Z.

Benore-Parsons, Marilee

Smith, Shelia R.

Stemmler, Timothy L.

Wasiukanis, Kristen

PubMed

Digital Commons@Wayne State University

Wayne State UniversityDigitalCommons@WayneStateBiochemistry and Molecular Biology FacultyPublications Department of Biochemistry and Molecular Biology1-1-2008Association of Copper to Riboflavin BindingProtein; Characterization by EPR and XASShelia R. SmithUniversity of Michigan - DearbornKrisztina Z. BenczeWayne State UniversityKristen WasiukanisWayne State UniversityTimothy L. StemmlerWayne State University, tstemmle@med.wayne.eduMarilee Benore-ParsonsUniversity of Michigan - DearbornThis Article is brought to you for free and open access by the Department of Biochemistry and Molecular Biology at DigitalCommons@WayneState. Ithas been accepted for inclusion in Biochemistry and Molecular Biology Faculty Publications by an authorized administrator ofDigitalCommons@WayneState.Recommended CitationSmith SR, Bencze, KZ, Wasiukanis K, Stemmler TL, Benore-Parsons M. Open Inorg Chem J. 2008;2:22. doi:10.2174/1874098700802010022Available at: http://digitalcommons.wayne.edu/med_biochem/1522 The Open Inorganic Chemistry Journal, 2008, 2, 22-24    1874-0987/08 2008 Bentham Science Publishers Ltd. Association of Copper to Riboflavin Binding Protein; Characterization by EPR and XAS  Sheila R. Smith*,a, Krisztina Z. Benczeb, Kristen Wasiukanisa, Timothy L. Stemmler*,b and Marilee Benore-Parsonsa aDepartment of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48101,USA bDepartment of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, MI 49201, USA Abstract: The association of copper to Riboflavin Binding Protein (RBP) from egg white has been studied by electron paramagnetic resonance (EPR) and X-ray absorption (XAS) spectroscopies. The type II site contains a mix of copper I and II in an oxygen rich environment. Keywords: Riboflavin binding protein, copper, gallus-gallus.  The association of copper to Riboflavin Binding Protein (RBP) from egg white has been studied by electron par-amagnetic resonance (EPR) and X-ray absorption (XAS) spectroscopies in order to provide insight into how this es-sential protein may transport and store copper in avian em-bryos. Riboflavin Binding Protein, RBP, purified from avian egg white, has been shown to bind copper in a 1:1 molar ratio when dialyzed against copper(II) [1]. While the egg is a unique environment and quite rich in copper, the mecha-nisms by which this copper is delivered during development and stored for eventual use remain unclear [2]. Since RBP is already identified in the active transport of the cofactor ribo-flavin to the egg, evidence of its copper binding ability may suggest an additional role for RBP in the transport and stor-age of copper.    Copper binding to RBP is a recently identified and novel interaction associated with the protein [1]. As purified by the conventional method [3], RBP contains only trace amounts of copper. The loss of copper during purification and subse-quent failure to identify a copper binding ability in previous studies may be attributed to the somewhat extreme condi-tions applied to the protein during the standard purification, specifically to the necessity for pH 4 acetate buffer in several of the elution steps. Aerobic dialysis against buffer contain-ing cupric chloride affords a protein fully loaded with one equivalent of copper, a ratio that has remained fairly constant through various dialysis conditions.   EPR results confirm that the copper binding site is a well-ordered type II site, as shown in Fig. (1). Type II is de-fined as 4-5 coordinate copper bound to a mixture of N, O and/or S ligands. Spectral parameters unique to this system are: g=2.06 (A=10G) and g||=2.39 (A||=142 G) [1]; these spectral parameters were obtained by simulation, as outlined previously [1] using the XSOPHE software package of  *Address correspondence to these authors at the Department of Natural Sciences, University of Michigan-Dearborn, Dearborn, MI 48101,USA.Fax: 313-593-4937; Tel: 313-583-6399; E-mail: sheilars@umd.umich.edu Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, MI 49201, USA. Fax: 313-577-2765; Tel: 313-577-5712; E-mail: tstemmle@med.wayne.edu Bruker. Simulation results are consistent with a copper bind-ing site containing 3-4 oxygens in the first coordination sphere [4]. The small feature visible in the EPR spectrum at approximately 3600 G is an artifact associated with the cav-ity.                 Fig. (1). Continuous Wave (CW) EPR of copper loaded Riboflavin Binding Protein (1.65 mM RBP) in 0.10 mM TRIS, pH 7.2. Mi-crowave Frequency: 9.7 GHz. Microwave Power: 2.0 μW, Modula-tion Frequency: 10 kHz, Modulation Amplitude: 10 G, Tempera-ture: 10K.  XAS studies, performed on copper loaded RBP, were used to examine the bound metal’s oxidation state and metal-ligand coordination structure. A substantial pre-edge feature at ca. 8978 and the minor feature at 8984 eV, indicative of 1s3d and 1s4p electronic transitions typically seen for Cu(II) and Cu(I), respectively, suggest that while the bulk of the copper bound to RBP is Cu(II) a substantial distribution of Cu(I) is also bound to RBP (Fig. 2). EXAFS simulations Association of Copper to Riboflavin Binding Protein The Open Inorganic Chemistry Journal, 2008, Volume 2    23  (Fig. 3A), used to characterize the copper-nearest neighbor ligand structure, indicate an average coordination environ-ment constructed by three oxygen/nitrogen based ligands at an average bond distance of 1.96 Å. These data could not be fit with any copper-sulfur or copper•••copper scattering. The presence of carbon scattering at > 3Å, observed in the Fou-rier transform of the Cu EXAFS (Fig. 3B), indicates a rela-tive degree of long-range ligand order that is typically seen from carboxylate carbons or imidazole rings [5]. The final EXAFS fits table is provided as supplemental information.               Fig. (2). XANES of Cu(II) loaded RBP (top). Vertical dashed line indicates the 1s  4p transition at 8984 eV. Inset shows the expan-sion of the background-subtracted 1s 3d region of the XANES spectra.  Since photoreduction of copper is common in XAS, EPR spectroscopy was used to quantitate the relative amounts of copper(I) and copper(II) bound to the protein by comparison to copper sulfate standard samples. Spin quantitation shows that the protein, as loaded aerobically with copper II in the absence of external reducing agents contains between 30 and 50% copper I in various samples tested, suggesting that while some of the signal for copper I seen in the XANES data might be attributed to photoreduction, some reduction of the copper II is occuring as a result of the binding interac-tion.  The reduction of copper upon binding to RBP is an inter-esting result. Dialyses are performed on holo-RBP (RBP bound to its riboflavin cofactor) under aerobic conditions in the absence of any external reducing agents. Furthermore, a stable copper I site featuring no sulfur or histidyl ligands would be most unusual. One possible source of reducing equivalents from the protein would be the bound riboflavin moiety. Experiments are ongoing to identify the specific ligands to copper in the binding site and to identify the re-ducing agent, in addition to a modification of the purification protocol so that we might detect natively bound copper in the protein as purified.  Since RBP is known to be involved in the active trans-port of riboflavin in egg, and since the delivery system for copper into the egg is not fully understood, the implication of the current work is that RBP could participate in the ac-tive transport of copper into the egg. Furthermore, since RBP has been shown to be capable of reducing the copper, the protein may play some role in the stabilization of the copper redox state for bound metal. The possibility of a new copper transport protein that also modulates the redox properties of its substrate are of extreme interest, as copper metal is util-ized ubiquitously at different locations in the egg during all stages of embryonic development.              Fig. (3). EXAFS and Fourier transforms of copper loaded RBP XAS data. Raw EXAFS spectra in black (A) along with the corresponding Fourier transform (B). Simulations of EXAFS and Fourier transform data are shown in green. 24    The Open Inorganic Chemistry Journal, 2008, Volume 2 Smith et al.  ABBREVIATIONS RBP = Riboflavin Binding Protein EPR = Electron paramagnetic resonance XAS = X-ray absorption spectroscopy XANES = X-ray absorption near edge spectroscopy EXAFS = Extended X-ray absorption fine structure ACKNOWLEDGEMENTS  This work was supported by the National Institute of Diabetes and Digenstive and Kidney Diseases R01DK068139 (TLS). Special thanks to Professor John McCracken and the Michigan Center for Structural Biology for use of the EPR facilities. NOTES AND REFERENCES General Chemicals  TRIS, and CuCl2 were purchased from Sigma (St. Louis, MO).  Protein Purification  Purification and concentration quantification RBP from egg whites followed published protocols [3].  Dialysis for Copper Loading  Lyophilized Protein was re-suspended in 0.1 M pH 7.2 Tris and dialyzed overnight aerobically against Tris buffer containing 10 mM CuCl2. This dialysis was followed with dialysis against pure Tris buffer to remove any excess cop-per. Total copper was quantitated using graphite furnace atomic absorption spectroscopy as previously reported [1]. Protein is digested in a nitric acid solution, followed by the addition of 3% Hydrogen Peroxide. Copper is measured by atomic absorption spectroscopy by comparison a standard curve of analytical aqueous copper solutions.  EPR Spectroscopy  Continuous wave electron paramagnetic resonance stud-ies were performed at 10K on a Bruker E680X spectrometer equipped with an Oxford CF935 cryostat and either a 5mm dielectric resonator or a 4 mM dielectric resonator equipped with an ENDOR accessory.  XAS Experiments  XAS studies were carried out at both the Stanford Syn-chrotron Radiation Laboratory (SSRL), on beamline 9-3, and at the National Synchrotron Light Source (NSLS), on beam-line X9b. SSRL is a national user facility operated by Stan-ford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Mo-lecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the NIH, National Center for Research Resources, Biomedical Technology Program. NSLS, located at Brook-haven National Laboratory, is supported by the U.S. De-partment of Energy, Division of Materials Sciences and Di-vision of Chemical Sciences, under Contract No. DE-AC02-98CH10886. Spectra were collected on multiple reproduci-ble samples. Data collection and analysis protocols have been previously reported [6]. SUPPORTIVE/SUPPLEMENTARY MATERIAL  A table containing the final EXAFS fits is included as supplemental information. REFERENCES [1] Smith, S.R.; Pala, I.; Benore-Parsons, M. J. Inorg. Biochem., 2006, 100, 1730. [2] Richards, M.P. Poultry Sci., 1997, 76, 152. [3] Miller, M.S.; White III, H.B. Meth. Enzymol., 1986, 227. [4] Peisach, J.; Blumberg, W.E. Arch. Biochem. Biophys., 1974, 165, 691. [5] Hakemian, A.E.; Tinberg, C.E.; Kondapalli, K.C.; Tesler, J.; Hoffman, B.M.; Stemmler, T.L.; Rosenzweig, A.C. J. Am. Chem. Soc., 2005, 127, 17142. [6] Lieberman, R.L.; Kondapalli,K.C.; Shrestha, D.B.; Hakemian, A.S.; Smith, S. M.; Telser, J.; Kuzelka, J.; Gupta, R.; Borovik, A. S.; Lippard, S. J.; Hoffman, B. M.; Rosenzweig, A. C. ; Stemmler, T. L. Inorg. Chem., 2006, 45, 8372.    Received: January 02, 2008 Revised: February 22, 2008 Accepted: February 22, 2008   

Association of Copper to Riboflavin Binding Protein; Characterization by EPR and XAS

http://digitalcommons.wayne.edu/cgi/viewcontent.cgi?article=1000&context=med_biochem

Association of Copper to Riboflavin Binding Protein; Characterization by EPR and XAS

Abstract

Similar works

Full text

Available Versions

Digital Commons@Wayne State University