Research Network for Metals in Medicine

 

 

Professor Graeme Richard Hanson

BSc(Hons), PhD(LaTrobe)

Position: Senior research fellow

Affiliation: Centre for magentic resonance

Postal Address:
Centre for Magnetic Resonance
The University of Queensland
St. Lucia, Qld., 4072
AUSTRALIA

Phone: +61 (07) 3365 3242
Fax: +61 (07) 3365 4242
Email:Graeme.Hanson@cmr.uq.edu.au
Webpage: www.cmr.uq.edu.au


Research Profile

My major research interests include electron paramagnetic resonance spectroscopy (EPR) and its application to the characterisation of paramagnetic materials with special emphasis on the analysis (computer simulation) of continuous wave (CW) and pulsed EPR spectra, the metal binding sites in metalloproteins and transition metal ion complexes.

Specifically this has involved:

  • development of a computer simulation software suite (XSophe-Sophe-XeprView) for the analysis of continuous wave EPR spectra (XSophe 1.02, 1.04, 1.1, 1.1.2 and 1.1.3),
  • commercialisation of the XSophe computer simulation software through Bruker Biospin,
  • development of homotopy as an alternative approach for the computer simulation of randomly oriented EPR spectra,
  • development of an integrated computer simulation software suite (Molecular Sophe) based on molecular structure for the analysis of CW EPR, pulsed EPR, CW ENDOR and pulsed ENDOR spectra (Molecular Sophe 2.x).
  • structural characterisation of the molybdenum enzymes dimethylsulfoxide reductase and dimethylsulfide dehydrogenase.
  • structural characterisation of the binuclear Fe(III)-m-(OH)-Zn(II) and Fe(III)-m(O)-Mn(II) centres in red kidney bean and sweet potato purple acid phosphatases.
  • structural characterisation of human isoenzymes of cytochrome P450 and its substrate complexes employing CW and pulsed EPR spectroscopy.
  • structural characterisation of mono- and bi-nuclear copper(II) complexes with cyclic peptides.
  • characterisation of mono- and poly-nuclear nitroxides using CW and pulsed EPR spectroscopy and
  • characterisation of vanadium insulin enhancing drugs, their transport and metabolism.

Summary of Expertise
I am committed to maintaining a world class EPR facility and providing expertise in EPR spectroscopy to users within and outside the University of Queensland. Towards this end my group have:

  • during the last two years, undertaken the development of an integrated approach for the computer simulation of continuous wave and pulsed EPR and ENDOR spectra, energy level diagrams, transition roadmaps and transition surfaces. This approach, based on molecular structure, will revolutionise the 3-dimensional molecular characterisation of paramagnetic materials using EPR spectroscopy as until now the analysis of complex CW and pulsed EPR spectra has been based on a spin system rather than molecular structure. The approach employing object oriented programming has involved the development of a:
    • completely new X-windows interface (XSophe) written in C++ and employing the GUI builder BxPro,
    • general C++ parser allowing the input/output of spectral, spin Hamiltonian and structural parameters and enabling the expansion of experiments (pulse sequences) to be easily integrated in the future,
    • C++ version of Sophe for the analysis of CW and pulsed EPR and ENDOR spectra. This software has been based around the SOPHE grid (patented) and has employed the mosaic misorientation linewidth model, frequency domain pulsed simulations, Floquet theory and distributions of spin Hamiltonian and structural (internuclear distances and orientations) parameters.
    • extended the XSophe-Sophe-XeprView computer simulation software suite for the analysis of continuous wave EPR spectra. There are two patents associated with this research which is now being performed collaboratively with the EPR Division of Bruker Biospin (Germany). The XSophe software suite is sold by Bruker at a cost of 6,200 Euro / license together with a software maintenance agreement. The software suite is now available on SGI IRIX (6.2, 6.3, 6.4, 6.5) and Linux platforms (RedHat, 6.2, 7.1, 7.2, 7.3, 8.0, 9.0; Mandrake 8.1, 8.2, 9.0, 9.1) and Versions 1.02, 1.04, 1.1, 1.1.2 and 1.1.3 (April, 2003) have been released. The latest version (1.1.x) of the software includes energy level diagrams, transition roadmaps, transition surfaces, bug fixes and new installation scripts allowing operating system upgrades to be performed easily. An anonymous ftp (ftp and sftp) server has been established within CMR so that the software can be uploaded by users as soon as new releases become available.
    • identified the presence of sulfur centered radicals upon reduction of dimethylsulfoxide reductase (DMSOR) with sodium dithionite using CW and pulsed EPR spectroscopy. The formation of these centres can occur through intramolecular electron transfer of the Mo(VI) and Mo(V) centres to form an S=1 Mo(V) (P-MGD) (Q-MGD: S-C=C-S-) (MGD-molybdopterin guanine dinucleotide) moeity which undergoes coupled proton electron transfer to form the Mo(IV) (P-MGD) (Q-MGD: S-C=C-S-) centre. Hyperfine sublevel correlation spectroscopy (HYSCORE) reveals that the unpaired electron is delocalised onto N-8 of the pyranopterin of the Q-MGD.
    • employed variable temperature (120-2K) X-band EPR spectroscopy to characterise the multiple redox centres, Mo(V), [3Fe-4S]+, [4Fe-4S]+ in 'as isolated' dimethylsulfide dehydrogenase. A pH dependent EPR study of the Mo(V) centre in 1H2O and 2H2O reveals the presence of three Mo(V) species in equilibrium, Mo(V)-OH2, Mo(V)-X and Mo(V)-OH. Between pH6 and 8.2 the dominant species is Mo(V)-OH2 and Mo(V)-X is a minor component. X is probably the anion, chloride. Comparison of the rhombicity and anisotropy parameters for the Mo(V) species in DMS dehydrogenase with other Mo(V) centres in metalloproteins showed that it was most similar to the low pH nitrite spectrum of E. coli nitrate reductase (NarGHI). A [4Fe-4S]+ cluster was also identified with unusual spin Hamiltonian parameters (g1, 2.0158; g2, 1.8870; g3, 1.8620), suggesting that one of the iron atoms may have a fifth non-sulfur ligand. The g matrix for this cluster is very similar to that found for the minor conformation of Center 1 in NarH (Guigliarelli, B., Asso, M., More, C., Augher, V., Blasco, F., Pommier, J., Giodano, G., and Bertrand, P. (1992) Eur. J. Biochem. 307, 63-68). The two conformations in NarH may arise from an equilibrium involving the coordination/dissociation of a fifth ligating atom (N or O) to an Fe atom in the cluster. The minor conformation in NARH corresponds to the cluster in which the fifth ligand is coordinated. Analysis of a ddhC mutant showed that this gene encodes the b-type cytochrome in dimethylsulfide dehydrogenase. Magnetic circular dichroism studies revealed that the axial ligands to the iron in this cytochrome are histidine and methionine, consistent with predictions from protein sequence analysis. Redox potentiometry showed that the b-type cytochrome has a high mid-point redox potential (Eo = +315 mV, pH 8).
    • phylogenetic studies have shown that dimethylsulfide dehydrogenase, selenate reductase and E. coli nitrate reductase form a distinct class of oxomolybdenum enzymes. Whilst CW EPR studies have shown that the Mo ion is coordinated by 4-thiolate sulfur atoms from two pterins, and an aqua ligand. The protein side chain ligand has yet to be identified, though sequence homology suggests it is either Ser195, Thr214, or His220. Recent orientation selective pulsed HYSCORE studies have shown unambiguously that His220 is located in the second coordination sphere of the Mo ion.
    • shown that the purple acid phosphatase from sweet potato is the first reported example of an enzyme containing binuclear Fe-Mn centres. Multifield saturation magnetization data over a temperature range from 2 to 200 K indicates that these centres are strongly antiferromagnetically coupled. Metal ion analysis shows an excess of Fe over Mn. Low temperature EPR spectra reveal only resonances characteristic of high spin Fe(III) centres (Fe(III)-apo and Fe(III)-Zn(II)) and Cu(II). There were no resonances from either Mn(II) or binuclear Fe-Mn centres. Oxidation and reduction of the enzyme indicated that homobinuclear metal centres (Fe(III)-Fe(III) , Mn(III)-Mn(III) and Mn(II)-Mn(II)) were not present in the enzyme. Together with a comparison of spectral properties and sequence homologies between known purple acid phosphatases the spectroscopic data strongly indicate the presence of Fe(III)-Mn(II) centres in the active site of the sweet potato enzyme. Due to the strong antiferromagnetism it is likely that the metal ions in the sweet potato enzyme are linked via a µ-oxo bridge, in contrast to other known purple acid phosphatases where a µ-hydroxo bridge is present. Differences in metal ion composition and bridging may affect substrate specificities and hence the biological function of different purple acid phosphatases.
    • implemented Homotopy into the XSophe-Sophe electron paramagnetic resonance computer simulation suite, wherein it is used to find the eigenvalues and associated eigenvectors of the special class of matrices generated in the computer simulation of magnetic resonance spectra. In particular, by directly tracing the eigenfunctions in parameter space it can:
    • trace a given transition as a function of orientation (q = 0 -> 180o, f =0 -> 180o) in the presence of energy level anti-crossing,
    • trace looping transitions and
    • perform the simulations in frequency space. Since this method is also computationally faster than matrix diagonalisation, when combined with the Mosaic Misorientation linewidth model should result in significant reductions in computational time for the simulation of EPR spectra without sacrificing accuracy. In conjunction with the Sophe partition scheme homotopy improves the quality of simulated spectra, allows the analysis of more complicated EPR spectra from complex spin systems and reduces the computational time compared with matrix diagonalisation. The method can also be applied to ENDOR, electron spin echo envelope modulation (ESEEM), solid state nuclear magnetic resonance and nuclear quadrupole resonance spectra.
    • in conjunction with Dr. Lutz Lötzbeyer from Prof. Peter Comba's laboratory at the University of Heidelberg and Dr. Rodney Cusack EPR and NMR studies were used to structurally characterise mono- and bi-nuclear copper(II), calcium(II) and zinc(II) complexes with cyclic peptides.
    • in conjunction with Dr. Steven Bottle (Queensland University of Technology) and Dr. Duncan Gillies (University of Surrey) variable temperature CW and pulsed EPR has been employed to characterise a range of bis- and tris-nitroxides. The magnitude of the exchange coupling between the electron spins is similar to that of the nitrogen hyperfine coupling and applying the EXSY pulse sequence has allowed the determination of the exchange coupling constant from the cross peaks.


Selected Publications

  1. Bottle, S.E.; Hanson, G.R.; Micallef, A.S., “Application of the new EPR spin trap 1,1,3-trimethylisoindole N-oxide (TMINO) in trapping HO. and related biologically important radicals”, Org. Biomolec. Chem., 2003, 2585-2589.
  2. Hanson, G.R.; Gates, K.E.; Noble, C.J.; Mitchell, A.; Benson,S.; Griffin, M.; Burrage, K., "XSophe - Sophe - XeprView A Computer Simulation Software Suite for the Analysis of Continuous Wave EPR spectra", in EPR of Free Radicals in Solids: Trends in Methods and Applications, Shiotani, M.; Lund, A. (Eds.), Kluwer Press. In Press, 2003, pp197-237.
  3. McDevitt C.A.; Hanson, G.R.; Noble C.J.; Cheesman M.R.; McEwan A.G. "Characterization of the redox centers in Dimethylsulfide Dehydrogenase from Rhodovulum sulfidophilum", Biochemistry, 2002, 41, 15234 -15244.
  4. Bernhardt, P.V; Comba , P.; Fairlie, D.P.; Gahan, L.R.; Hanson, G.R.; Lötzbeyer, L. "Synthesis and structural properties of patellamide A derivatives and their copper(II) compounds", Chem. Eur. J., 2002, 8, 1527-1536.
  5. Schenk, G.; Boutchard, C.L.; Carrington, L. E.; Noble, C.J.; Moubaraki, B.; de Jersey, J.; Hanson, G.R.; Hamilton, S. "A purple acid phosphatase from sweet potato contains an antiferromagnetically coupled binuclear Fe-Mn centre", J. Biol. Chem., 2001, 276, 19084-19088.
  6. Bell, A.F.; He, X.; Ridge, J.P.; Hanson, G.R.; McEwan, A.G.; Tonge, P.J."Active Site Heterogeneity in Dimethyl Sulfoxide Reductase from Rhodobacter capsulatus Revealed by Raman Spectroscopy" Biochemistry, 2001, 40, 440-448.
  7. Huang, X.; Cuajungeo, M.P.; Atwood, C.S.; Hartshorn, M.A.; Tyndall, J.D.A.; Hanson, G.R.;Stokes, K.C.; Leopold, M.; Multhaup, G.; Goldstein, L.E.; Scarpa, R.C.; Saunders, A.J.; Lim, J.; Moir, R.D.; Glabe, C.; Bowden, E.F; Masters, C.L.; Fairlie, D.P.; Tanzi, R.E.; Bush, A.I. "Cu(II) Potentiation of Alzheimer Ab Neurotoxicity-Correlation with Cell-Free Hydrogen Peroxide Production and Metal Reduction", J. Biol. Chem., 1999, 274, 37111-37116.
  8. Griffin, M.; Muys, A.; Noble, C.; Wang, D.; Eldershaw, C.; Gates, K.E.; Burrage, K.; Hanson, G.R."XSophe, a Computer Simulation Software Suite for the Analysis of Electron Paramagnetic Resonance Spectra", 1999, Mol. Phys. Rep., 26, 60-84.
  9. Comba, P.; Cusack, R; Fairlie, D.P.; Gahan, L.R.; Hanson, G.R.; Kazmaier, U.; Ramlow, A. "The Solution Structure of a Copper(II) Compound of A New Cyclic Octapeptide by EPR Spectroscopy and Force Field Calculations", Inorg. Chem., 1998; 37, 6721-6727.
  10. Baugh, P.E.; Garner, C.D.; Charnock, J.M.; Collison, D.; Davies, E.S.; McAlpine, A.S.; Bailey, S.; Lane, I.; Hanson, G.R.; McEwan, A.G. "X-ray Absorption Spectroscopy of Dimethylsulfoxide Reductase from Rhodobacter capsulatus", J. Biol. Inorg. Chem., 1997, 634-643.

  11. Patents
  12. Wang, D.; Hanson, G.R. "Computer Simulation of Magnetic Resonance Spectra", Australian Patent, 1996, No. 726393, PCT/AU96/00534, WO 97/08630
  13. Gates, K.E.; Hanson, G.R.; Burrage, K. "Application of Homotopy in the Analysis of Magnetic Resonance Spectra", Australian Patent, 1997, No. 721790.
  14. Gates, K.E.; Hanson, G.R.; Burrage, K. "Application of Homotopy in the Analysis of Magnetic Resonance Spectra", United Kingdom Patent, 1997, No. 2320579.
  15. Wang, D.; Hanson, G.R. "Computer Simulation of Magnetic Resonance Spectra", US Patent, 1996, No. 6,236,202.
  16. Gates, K.E.; Hanson, G.R.; Burrage, K. "Application of Homotopy in the Analysis of Magnetic Resonance Spectra", US Patent, 1997, No. 6,052,519.

Facilities

Multifrequency (Q-, X-, S-band) Variable temperature (1.5-700K) Continuous Wave Bruker Elexsys E500 EPR Spectrometer

The multifrequency continuous wave EPR Spectrometer available in CMR is equiped with Q-band (35 GHz), X-band (9GHz) and S-band ( 4 GHz) microwave bridges and the following resonators (Q-band: clylindrical TE011, X-band: Rectangular TE102, Double rectangular TE104 (spin quantitation expts.), Dual mode (perpendicular and parallel mode cavity for studying even electron spin systems), Super high Q cavity (for measuring 10-9 M radicals), Optical cavity for irradiating samples and an S-band Flexline resonator. The spectrometer is equipped with a 10” electromagnet with a 12 kW power supply providing a maximum field strength of 1.5 T. This can be extended to 1.83T with high field pole tips. Variable temperature capabilities (1.9 K - 700 K) for all three microwave frequencies. Low temperature (1.9-77 K) measurements are performed using the Oxford Instruments ESR-910 and the CF-935 cryostats in conjunction with an Oxford Instruments ITC-4 temperature controller. Measurements above 77 K employ either a nitrogen boil off system or nitrogen gas in conjunction with a Eurotherm B-VT-2000 temperature controller. Calibration of the Magnetic Field (Bruker ER035M Gaussmeter) and Microwave Frequency (EIP 548B Microwave Frequency Counter). Rapid scan capabilities and fast digitiser (A/D conversion time 50 nsec) capabilities. An optical system - 1000 W Hg/Xe lamp. This can be used at either X- or Q-band microwave frequencies and at all temperatures. A variety of wavelength cutoff filters are available. The spectrometer is equipped with a manual and automatic single circle goniometer for single crystal measurements and is ethernet linked to local area network and the world.

Continuous Wave and Pulsed EPR, END(T)OR and ELDOR X-band
Variable temperature (1.5-700K) Bruker Elexsys E580 EPR Spectrometer

Elucidation of three dimensional crystallographic information (distance and orientation) for a paramagnetic centre relies on the observation of hyperfine coupling between nuclei and electron spins which is often unresolved in randomly orientated continuous wave EPR spectra. The advent of multidimensional pulsed EPR, electron nuclear double (triple) resonance (END(T)OR) spectroscopy in conjunction with orientation selective experiments and computer simulation overcomes this problem and allows three dimensional structures of paramagnetic centres to be determined.

The Bruker Elexsys E580 CW & pulsed EPR spectrometer installed in May 2001, has CW and pulsed END(T)OR capabilities, a 10" magnet with a 12kW power supply and a heat exchanger, a teslameter (FT NMR spectrometer) and a microwave frequency counter for magnetic field and microwave frequency calibration respectively, a continuous flow (sub 4K) cryostat with an ITC 503 variable temperature controller and Aqua X cells for measuring aqueous samples. The spectrometer is unique in the southern hemisphere and the low temperature cryostat, is unique in the world allowing the characterisation of high spin centres, such as heme and non-heme iron proteins.

In conjunction with the multifrequency CW EPR spectrometer and the XSophe computer simulation software suite the EPR facilities are unique in the southern hemisphere and allow Australian scientists to structurally characterise paramagnetic molecules such as high Tc superconductors, organic molecular ferromagnets, catalysts, free radicals, transition metal ion complexes and metalloproteins found in such diverse areas as solid state physics, geology, materials science, inorganic, organic and polymer chemistry, biochemistry, pharmacy, food science, radiation dosimetry and medicine.




The Xsophe-Sophe-XeprView Computer Simulation Software Suite

The XSophe-Sophe-XeprView computer simulation software suite enables scientists to easily determine spin Hamiltonian parameters from isotropic, randomly oriented and single crystal continuous wave electron paramagnetic resonance (CW EPR) spectra from radicals and isolated paramagnetic metal ion centers or clusters found in metalloproteins, chemical systems and materials science. XSophe provides an X Windows graphical user interface to the Sophe programme and allows: creation of multiple input files, local and remote execution of Sophe, the display of sophelog (output from Sophe) and input parameters/files. Sophe is a sophisticated computer simulation software programme employing a number of innovative technologies including; the Sydney OPera HousE (SOPHE) partition and interpolation schemes, a field segmentation algorithm, the mosaic misorientation line width model, parallelization and spectral optimisation. In conjunction with the SOPHE partition scheme and the field segmentation algorithm, the SOPHE interpolation scheme and the mosaic misorientation linewidth model greatly increase the speed of simulations for most spin systems. Employing brute force matrix diagonalization in the simulation of an EPR spectrum from a high spin Cr(III) complex with the spin Hamiltonian parameters ge = 2.00, D = 0.10 cm-1, E/D = 0.25, Ax =120.0, Ay = 120, Az = 240 x 10-4 cm-1 requires a SOPHE grid size of N=400 (to produce a good signal to noise ratio) and takes 229.47 sec. In contrast the use of either the SOPHE interpolation scheme or the mosaic misorientation linewidth model requires a SOPHE grid size of only N= 18 and takes 44.08 sec. and 0.79 sec. respectively. Results from Sophe are transferred via the Common Object Request Broker Architecture (CORBA) to XSophe and subsequently to XeprView where the simulated CW EPR spectra (1D and 2D) can be compared to the experimental spectra. Energy level diagrams, transition roadmaps and transition surfaces aid the interpretation of complicated randomly oriented CW EPR spectra and can be viewed with a web browser and an OpenInventor scene graph viewer.



Rapid Freeze Quench Instrument
The rapid freeze-quench facility will make it possible to trap reaction intermediates on the millisecond time-scale and allow their spectroscopic properties to be studied. The equipment consists of precision mixing lines, reaction chamber, stepper motor pumps together with micro-droplet ejection nozzle into a cryogenic trapping matrix. The study of such intermediates provides the key to understanding important biochemical reaction mechanisms. The facility allows structural information to be gained on the intermediate species through the existing UQ world class EPR and MCD spectroscopic facilities. Because this type of information is not possible from crystallographic studies, the rapid freeze – quench technique is a rapidly emerging technology in the field of structural biology.

International Linkages

Bruker Biospin, Germany - Software development and commercialisation.
Prof. Peter Comba, University of Heidelberg, Germany – Binuclear Copper(II) complxes and their interaction with cyclic peptides.
Prof. Chris Orvig, University of British Columbia, Canada – Vanadium Insulin Enhancing Drugs.
Prof. Wolfgang Lubitz, Max Planck Institute for Bioinorganic Chemistry, Germany – EPR of metalloproteins
Dr. Frank Neese, Max Planck Institute for Bioinorganic Chemistry, Germany – Computational calculations (interface between XSophe and DFT software)