INVESTIGATION OF THE PARAMAGNETIC DOMAIN OF PUTIDAREDOXIN BY N-15 NMR-SPECTROSCOPY

The oxidized forms of the redox proteins putidaredoxin and putidaredox in-N-15 were prepared by insertion of suitable plasmids into E. coli a nd the structures of the proteins were studied by home-and heteronucle ar H-1 and N-15 NMR methods, including identification of N-15 resonanc e types by one-and two-dimensional DEFT spectrum editing and measureme nts of N-15 chemical shifts, coupling constants, nuclear Overhauser ef fects and spin-lattice relaxation times. Direct detection of N-15 NMR spectra revealed a number of N-15 resonances that have not been found in previous H-1 detected NMR studies. These resonances include a set o f 16 paramagnetically broadened signals from N-15 nuclei that are stru cturally close to the iron-sulfur cluster of the protein, proline back bone N-15 resonances and N-15 signals from the side-chains of glutamin e and Iysine residues. Reduced distances between certain paramagnetica lly affected N-15 nuclei and the center of the iron-sulfur cluster wer e calculated from the N-15 spin-lattice relaxation times, based on the assumption of a dominant electron-nuclear dipole-dipole relaxation me chanism. Ranked by size, these distances agree with those computed fro m coordinates of a published structure of putidaredoxin determined by H-1 NMR and analogy with an Anabaena ferredoxin. (C) 1997 John Wiley & Sons, Ltd.