The IH electron-nuclear double resonance (ENDOR) spectra in frozen buffer solutions of the reduced [2Fe-2S] clusters in adrenodoxin (Adx) and in the "Rieske" iron-sulfur protein (ISP) from the bovine mitochondrial bcl complex were measured at low temperatures (5-20 K) and analyzed by spectra reconstruction. A single paramagnetic species with iron valence states (II) and (III) connected uniquely to the cluster irons was found in both proteins. For Adx, the experimental spectra from 23 ¡ positions across the nearly axial (gm~x = 2.0241, gint -----1.9347, and gro,, = 1.9331) electron paramagnetic resonance (EPR) spectrum were analyzed. Four larger hyperfine couplings were assigned to the cysteine [3-protons near the Fe(III) ion. Transfer into the crystal structure showed that the Fe(III) ion was coordinated to the residues Cys55 and Cys92. The spin density was estimated as + 1.60 for the Fe(III) and -0.6 for the Fe(II) ion, respectively. The g-tensor direction with respect to the cluster showed strong similarities with the earlier assignment in Arthospira platensis ferredoxin (Canne C., Ebelshauser M., Gay E., Shergill J.K., Cammack R., Kappl R., Hª J.: J. Biol. Inorg. Chem. 5, 514, 2000). Ah Adx mutant (T54A) exhibiting a change (70 mV) in redox potential showed no significant influence at the [2Fe-2S] cluster. The Rieske ISP was subjected to the same analysis. The ENDOR spectra from 35 field positions across the rhombic (gmax = 2.028, gint = 1.891, and gm~, = 1.757) EPR spectrum were simulated. Three major proton contributions were identified from the o¡ behavior. Two were assigned to cysteine ~-protons and one to a ~-proton of Hisl41. In contrast to Adx, the direction of the gm,~-component was found to lie roughly in the FeScore plane and the largest proton coupling occurred along g~,t. The spin population was estimated as about +1.6 for the oxidized and -0.55 for the reduced iron.

The 1H electron nuclear double resonance (ENDOR) spectra in frozen solutions of the reduced [2Fe-2S] cluster in ferredoxin from Arthrospira (Spirulina) platensis have been measured at low temperatures (5-20 K) and simulated using orientational selection methods. The analysis confirmed the existence of a single paramagnetic species with iron valence states II and III connected uniquely to the cluster irons. The experimental ENDOR spectra were fitted to a model including the spin distribution on the centre, the orientation of the g-matrix, and the isotropic and anisotropic hyperfine couplings of the nearest protons in the crystallographically determined structure. In order to partially simulate ENDOR line shapes, a statistical distribution of the corresponding torsion angles between the Fe(III) centre and one of the beta-CH2 protons was introduced. From the analysis, four of the larger hyperfine couplings found were assigned to the cysteine beta-protons near the Fe(III) ion of the cluster, with isotropic hyperfine couplings ranging from 1.6 to 4.1 MHz. The spin distribution on the two iron ions was estimated to be +1.85 for the Fe(III) ion and -0.9 for the Fe(II) ion. The Fe(III) ion was identified as being coordinated to the cysteine ligands Cys49 and Cys79, confirming previous NMR results. The direction of the g-tensor with respect to the cluster was deduced. The g1-g2 plane is parallel to the planes through each iron and its adjacent cysteine sulfurs; the g2-g3 plane is nearly perpendicular to the latter planes and deviates by 25 degrees from the FeSSFe plane. The g1 direction is dominated by the bonding geometry of Fe(II) and does not align with the Fe(II)-Fe(III) vector.