STRUCTURAL DETERMINATION OF PARAMAGNETIC CENTER IN PUTIDAREDOXIN

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Structural determination of paramagnetic proteins by solid state and solution NMR techniques is limited by severe line broadening due to the strong hyperfine interaction between the nuclei and the paramagnetic center. This prevents the use of standard NMR structural methods within a few angstroms of the metal center. In this context, high frequency (140 GHz) pulsed ENDOR techniques are ideally suited to extract hyperfine interactions in the vicinity of the paramagnetic center. We have been investigating a 2-Fe 2-S ferredoxin putidaredoxin (Pdx), where the metal center displays an effective spin S = 1/2 in the reduced form (Fe" - Fe") and the cluster is ligated by four cysteinyl sulfurs. We were able to record 140 GHz spin-echo detected spectra at low temperatures (10 K) with excellent signal-to-noise (> 50) and determine the g-anisotropy to g,, = 2.02 and g_L = 1.94. TI-relaxation of the Fe-cluster was measured between 9 and 40 K. We found short T,- values of 50 gs at 9 K and a more complex biexponential behavior between 15 - 40 K, likely due to the thermal population of higher excited spin states. A special ENDOR resonator for efficient irradiation of low-gamma nuclei was developed and preliminary "C-Mims-ENDOR spectra were recorded at 10 K. In contrast to ENDOR data previously reported in the literature at low EPR frequencies (35 GHz), the ' 3C larmor frequency at 5 Tesla (53 MHz) is well separated from other nuclei and we observe symmetric ENDOR lineshapes around the larmor frequency. In a "C uniformly labeled protein, two distinct hyperfine splitting have been observed, which we attribute to the cystein methylene groups. Further experiments with selectively labeled proteins are in progress and will permit unambiguous assignments of the observed hyperfine couplings.