Mossbauer and electron paramagnetic resonance studies of the cytochrome bfcomplex

The Fe-57-enriched cytochrome bf complex has been isolated from hydrocultur es of spinach. It has been studied at different redox states by optical, EP R, and Mossbauer spectroscopy. The Mossbauer spectrum of the native complex at 190 K with all iron centers in the oxidized state reveals the presence of four different iron sites: low-spin ferric iron in cytochrome b [with an isomer shift (delta) of 0.20 mm/s, a quadrupole splitting (Delta E-Q) of 1 .77 mm/s, and a relative area of 40%], low-spin ferric iron of cytochrome f (delta = 0.26 mm/s, Delta E-Q = 1.90 mm/s, and a relative area of 20%), an d two high-spin ferric iron sites of the Rieske iron-sulfur protein (ISP) w ith a bis-cysteine and a bis-histidine ligated iron (delta(1) = 0.15 mm/s, Delta E-Q1 = 0.70 mm/s, and a relative area of 20%, and delta(2) = 0.25 mm/ s, Delta E-Q2 = 0.90 mm/s, and a relative area of 20%, respectively). EPR a nd magnetic Mossbauer measurements at low temperatures corroborate these re sults. A crystal-field analysis of the EPR data and of the magnetic Mossbau er data yields estimates for the g-tensors (g(z), g(y), and g(x)) of cytoch rome b (3.60, 1.35, and 1.1) and of cytochrome f (3.51, 1.69, and 0.9). Add ition of ascorbate reduces not only the iron of cytochrome f to the ferrous low-spin state (delta = 0.43 mm/s, Delta E-Q = 1.12 mm/s at 4.2 K) but als o the bis-histidine coordinated iron of the Rieske 2Fe-2S center to the fer rous high-spin state (delta(2) = 0.73 mm/s, Delta E-Q2 = -2.95 mm/s at 4.2 K). At this redox step, the Mossbauer parameters of cytochrome b have not c hanged, indicating that the redox changes of cytochrome f and the Rieske pr otein did not change the first ligand sphere of the low-spin ferric iron in cytochrome b. Reduction with dithionite further reduces the two hemes of c ytochrome b to the ferrous low-spin state (delta = 0.49 mm/s, Delta E-Q 1.0 8 mm/s at 4.2 K). The spin Hamiltonian analysis of the magnetic Mossbauer s pectra at 4.2 K yields hyperfine parameters of the reduced Rieske 2Fe-2S ce nter in the cytochrome bf complex which are very similar to those reported for the Rieske center from Thermus thermophilus [Fee, J. A., Findling, K. L ., Yoshida, T., et al. (1984) J. Biol. Chem. 259, 124-133].