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Current Research Interests
Mechanisms of Bioenergetic Membrane Proteins
&
Modern EPR Spectroscopy
Much of my recent research in Frankfurt has been supported by a site-grant from the German Research Council (DFG) entitled
SFB 472 - Molecular Bioenergetics in the project section P15:Prisner/MacMillan
"Multi-dimensional EPR-Spectroscopy of protein-bound redox-active
paramagnetic centres"
Overview
The aim of this project section is to investigate the functional role of paramagnetic centres in
electron-transfer (ET) and catalytic reactions of membrane protein complexes (e.g. in the respiratory chain, see figure).
EPR-Spectroscopy is an excellent method to characterise these centres and their local protein environment.
These paramagnetic centres can be separated into three classes, depending on their EPR characteristics :
Equipment
In the Frankfurt research group we have pulsed and
cw-EPR spectrometers working at 0,1 T (3 GHz, S-band), 0,3 T (9 GHz, X-band) and at 6,4 T (180 GHz, G-band).
The pulsed S- and G-band spectrometers have been developed and built in the group. cw- and pulsed-ENDOR experiments can be
performed at both X- anf G-band, PELDOR-methods have also been developed in the group at S- and X-band.
It is the combination of these specialised EPR-methods together with information from X-ray crystallographic data and other
spectroscopic techniques whcih allow a detailled description of these paramagnetic centres and their function.
Literature
A list of my recent publications involving these
topics is available
Transient organic radicals (e.g. semiquinones, chlorophylls, tyrosines, flavins) are short-lived intermediates in ET-reactions
and thus often only exist, under steady state conditions in proteins, in very low yields, making direct observation by
EPR impossible. They may however be detected by using time-resolved pulse EPR measurements, freeze-quenching after reaction
initiation or by altering the redox conditions. In order to differentiate between radical species and for a detailed
characterisation of their binding properties high-field EPR (B > 3T), ENDOR- (Electron Nuclear Double Resonance) and
pulse EPR methods are necessary. In many cases these species have long relaxation times so that 1- and 2- dimensional
pulse EPR methods, like ESEEM (Electron Spin Echo Envelope Modulation), HYSCORE (HYperfine Sublevel CORrElation spectroscopy)
and PELDOR (Pulsed ELectron DOuble Resonance) can be successfully applied to investigate the protein surrounding.
Protein-bound transition metals (e.g. Mn, Cu, Mo), which have either a structural function or are
often directly involved in ET- and catalytic redox reactions, can be studied with EPR spectroscopy in their paramagnetic states.
The spectra which one obtains are often complex and contain, in addition to the hyperfine couplings of ligands (and even of the
metal itself), information about spin-orbit coupling, their charge character, the ligand field as well as the covalency of the
ligand bonds, all of which directly influence the anisotropic G-tensor. In order to distinguish between these effects multi-frequency
EPR is especially important as the different contributions can be distinguished by their dependence on the external magnetic field.
As opposed to organic radicals, transition metal ions can be studied at standard magnetic fields of 0,3 T (X-band EPR) where orientation
selective experiments on powder samples can often be performed, assuming that the much shorter relaxation times allow this.
Thus structural information (distances and angles) can be determined.
Iron complexes (e.g. iron-sulphur complexes (FeS) and haem groups) constitute a special class of
paramagnetic centres in proteins for EPR spectroscopy. They are characterised by having very short relaxation times, so that
they are only observable at very low temperatures (<60 K). Hyperfine interactions are rarely observed in cw-EPR spectra, can
however be resolved using pulse EPR at very low temperatures (T<5 K). The fast relaxation of these iron species can however,
be used to obtain structural information on paramagnetic centres of the other two groups mentioned above (up to a distance of 1-3 nm),
and as these complexes are quite often part of the ET-chain of membrane proteins, this is often the case. In these cases the neighbouring
iron complex will directly influence the relaxation behaviour of the slower relaxing paramagnetic centres. Pulse EPR measurements of
these relaxation rates at different temperatures can provide the distance and relative orientation of the two paramagnetic molecules
within the protein.
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