Research Staff: Professor Graeme Richard Hanson

PROFESSORIAL RESEARCH FELLOW

Address:
Centre for Magnetic Resonance
Level 2, Gehrmann Laboratories
Research Road
The University of Queensland
Brisbane. QLD. 4072
E-mail address: Graeme Hanson
Telephone: +61-7-3365 3242
Fax: +61-7-3365 3833
Home Page: Graeme Hanson

Career Profile:
B.Sc.(Hons), Latrobe University, 1978
Ph.D., Latrobe University, 1984.

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

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, homotopy, 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 g_e = 2.00, D = 0.10 cm^-1, E/D = 0.25, A_x =120.0, A_y = 120, A_z = 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. The XSophe-Sophe-XeprView computer simulation software suite is avalaible from Bruker Biospin Germany.

Molecular Sophe Computer Simulation Software Suite
Molecular Sophe is an integrated computer simulation software suite (XSophe-Sophe-Xepr) based on molecular structure for the analysis of CW EPR, pulsed EPR, CW ENDOR and pulsed ENDOR spectra, energy level diagrams, transition roadmaps and transition surfaces (Molecular Sophe). 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.

EPR Studies of Metalloproteins
Throughout my career I have employed multifrequency CW-EPR and pulsed EPR to characterise metal binding sites in molybdoenzymes (xanthine oxidase, DMSO reductase, DMS dehydrogenase), iron sulfur proteins (lactyl dehydratase, Giardia ferredoxin), metallo-substituted enzymes (carboxypeptidase A, phospholipase C) and marine cyclic octapeptides.

Molybdenum Enzymes
We have 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.

Variable temperature (120-2K) X-band EPR spectroscopy has been employed 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 (E_o = +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 ligated to the Mo ion. This represents the first example of an oxomolybdenum enzyme with Histidine (nitrogen) in the primary or secondary coordination.

Purple Acid Phosphatases
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.

Copper(II) Cyclic Peptide Complexes
We have structurally characterised a wide range of mono- and bi-nuclear copper(II) cyclice peptide complexes empoying multifrequency EPR spectroscopy, mass spectrometery, optical absorption spectroscopy and circular dichroism. We have also extended these studies to examine calcium(II) and zinc(II) binding.

Nitroxides/Nitrones
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.

Transition Metal Ion Complexes
Variable temperature multifrequency CW EPR spectroscopy in conjunction with computer simulation has been employed to characterise mono- and bi-nuclear high spin Fe(III), Mn(II) complexes, copper(II), silver(II), molybdenum(V), tungsten(V) and chromium(V) complexes.

Vanadium (IV) Insulin Enhancing Drugs
The interactions of apo-transferrin and albumin with BMOV were studied by CW EPR revealing important ramifications on the design and biological fate of vanadium chelates with potential as antidiabetic pharmaceuticals. EPR studies demonstrate that identical reaction products are produced regardless of whether a BMOV or vanadyl sulfate (VOSO₄) source is introduced into a solution of apo-transferrin. Further detailed study rules out the presence of a ternary ligand-vanadyl-transferrin complex proposed in earlier work (Willsky et al., (2001) J. Inorg. Biochem., 85, 33) Differences in reaction products are observed for the interactions of BMOV and VOSO4 with albumin. Unlike with transferrin, the formation of an adduct between albumin and BMOV is detected. EPR spectra of BMOV-albumin solutions indicate the presence of vanadyl ions bound in a unique manner not observed in a comparable solution of VOSO₄ and albumin. Presentation of chelated vanadyl ions precludes binding at the numerous non-specific sites; provision of a chelating ligand, however, to a solution of VOSO4 and albumin causes a redistribution of the bound vanadyl ions from one binding site to another with concomitant binding of a maltol to form a new ternary complex. An analysis of solution equilibria and a model system of BMOV with 1-methylimidazole lends further support to the adduct binding mode proposed for BMOV and albumin. The stability of adduct formation between BMOV and 1-methylimidazole was also measured by difference UV spectroscopy, yielding a formation constant of log K₁ = 4.5(1). This is the first report of an in vitro reactivity difference between VOSO₄ and BMOV and may in fact have bearing on the form of the vanadium metabolite delivered to body tissues. Serum protein binding of prospective insulin-enhancing vanadium compounds likely has a dramatic effect on pharmacokinetics, transport and delivery of active metabolites to target tissue.

Selected Publications:
I have published 89 papers in internationally recognised journals, presented 102 papers at various scientific conferences and have 5 patent applications. A selection of publications is shown below.