EXPLORATION OF THE STRUCTURAL ENVIRONMENT OF THE IRON-SULFUR CLUSTER IN PUTIDAREDOXIN BY N-15 NMR-SPECTROSCOPY OF SELECTIVELY LABELED CYSTEINE RESIDUES

Putidaredoxin is a di-iron protein whose paramagnetic region is not we ll characterized by H-1 detected NMR. We have studied the structure of this region in greater detail by directly observed N-15 NMR of oxidiz ed and reduced putidaredoxin preparations in which the six cysteine re sidues are selectively labeled with N-15. A new method for preparation of a stable form of reduced putidaredoxin has been developed for use in NMR. The N-15 NMR spectra of the oxidized and reduced forms are cha racteristically different, and we have measured and compared N-15 chem ical shifts, spin-lattice relaxation times (T-1), and chemical shift/t emperature dependences for both forms. Evidence for localized valencie s of the iron atoms in the reduced form is presented. From the N-15 T- 1 values of the oxidized form, reduced distances of the cysteine backb one N-15 nuclei from the center of the Fe2S2 cluster have been calcula ted. These distances are consistent with those calculated from X-ray c rystal structure data for five ferredoxins, and confirm the structural similarity of the Fe2S2 clusters in putidaredoxin and in these ferred oxins in the oxidized state. (C) 1998 Academic Press.