Multifrequency pulsed electron paramagnetic resonance on metalloproteins.
Metalloproteins often contain metal centers that are paramagnetic in some functional state of the protein; hence electron paramagnetic resonance (EPR) spectroscopy can be a powerful tool for studying protein structure and function. Dipolar spectroscopy allows the determination of the dipole-dipole interactions between metal centers in protein complexes, revealing the structural arrangement of different paramagnetic centers at distances of up to 8 nm. Hyperfine spectroscopy can be used to measure the interaction between an unpaired electron spin and nuclear spins within a distance of 0.8 nm; it therefore permits the characterization of the local structure of the paramagnetic center's ligand sphere with very high precision. In this Account, we review our laboratory's recent applications of both dipolar and hyperfine pulsed EPR methods to metalloproteins. We used pulsed dipolar relaxation methods to investigate the complex of cytochrome c and cytochrome c oxidase, a noncovalent protein-protein complex involved in mitochondrial electron-transfer reactions. Hyperfine sublevel correlation spectroscopy (HYSCORE) was used to study the ligand sphere of iron-sulfur clusters in complex I of the mitochondrial respiratory chain and substrate binding to the molybdenum enzyme polysulfide reductase. These examples demonstrate the potential of the two techniques; however, they also highlight the difficulties of data interpretation when several paramagnetic species with overlapping spectra are present in the protein. In such cases, further approaches and data are very useful to enhance the information content. Relaxation filtered hyperfine spectroscopy (REFINE) can be used to separate the individual components of overlapping paramagnetic species on the basis of differences in their longitudinal relaxation rates; it is applicable to any kind of pulsed hyperfine or dipolar spectroscopy. Here, we show that the spectra of the iron-sulfur clusters in complex I can be separated by this method, allowing us to obtain hyperfine (and dipolar) information from the individual species. Furthermore, performing pulsed EPR experiments at different magnetic fields is another important tool to disentangle the spectral components in such complex systems. Despite the fact that high magnetic fields do not usually lead to better spectral separation for metal centers, they provide additional information about the relative orientation of different paramagnetic centers. Our high-field EPR studies on cytochrome c oxidase reveal essential information regarding the structural arrangement of the binuclear Cu(A) center with respect to both the manganese ion within the enzyme and the cytochrome in the protein-protein complex with cytochrome c.
Cluster of Excellence Macromolecular Complexes, Goethe-University, Frankfurt am Main, Germany.
Sevdalina
S
Lyubenova
Thorsten
T
Maly
Klaus
K
Zwicker
Ulrich
U
Brandt
Bernd
B
Ludwig
Thomas
T
Prisner
D001426:Bacterial Proteins / Q000737:chemistry*; D003300:Copper / Q000737:chemistry; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D003576:Electron Transport Complex IV / Q000737:chemistry* / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry*; D007501:Iron / Q000737:chemistry; D008667:Metalloproteins / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D010231:Paracoccus denitrificans / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D025062:Yarrowia / Q000737:chemistry*
D001426:Bacterial Proteins; D005656:Fungal Proteins; D008667:Metalloproteins; D003300:Copper; D045304:Cytochromes c; D007501:Iron; D003576:Electron Transport Complex IV