Mössbauer and electron paramagnetic resonance studies of the cytochrome bf complex.
The (57)Fe-enriched cytochrome bf complex has been isolated from hydrocultures of spinach. It has been studied at different redox states by optical, EPR, and Mössbauer spectroscopy. The Mössbauer 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 (DeltaE(Q)) of 1.77 mm/s, and a relative area of 40%], low-spin ferric iron of cytochrome f (delta = 0.26 mm/s, DeltaE(Q) = 1.90 mm/s, and a relative area of 20%), and two high-spin ferric iron sites of the Rieske iron-sulfur protein (ISP) with a bis-cysteine and a bis-histidine ligated iron (delta(1) = 0.15 mm/s, DeltaE(Q1) = 0.70 mm/s, and a relative area of 20%, and delta(2) = 0.25 mm/s, DeltaE(Q2) = 0.90 mm/s, and a relative area of 20%, respectively). EPR and magnetic Mössbauer measurements at low temperatures corroborate these results. A crystal-field analysis of the EPR data and of the magnetic Mössbauer data yields estimates for the g-tensors (g(z)(), g(y)(), and g(x)()) of cytochrome b (3.60, 1.35, and 1.1) and of cytochrome f (3.51, 1.69, and 0.9). Addition of ascorbate reduces not only the iron of cytochrome f to the ferrous low-spin state (delta = 0.43 mm/s, DeltaE(Q) = 1.12 mm/s at 4.2 K) but also the bis-histidine coordinated iron of the Rieske 2Fe-2S center to the ferrous high-spin state (delta(2) = 0.73 mm/s, DeltaE(Q2) = -2.95 mm/s at 4.2 K). At this redox step, the Mössbauer parameters of cytochrome b have not changed, indicating that the redox changes of cytochrome f and the Rieske protein 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 cytochrome b to the ferrous low-spin state (delta = 0.49 mm/s, DeltaE(Q) = 1.08 mm/s at 4.2 K). The spin Hamiltonian analysis of the magnetic Mössbauer spectra at 4.2 K yields hyperfine parameters of the reduced Rieske 2Fe-2S center 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].
Institut für Physik, Medizinische Universität Lübeck, Germany.
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Haehnel
D001205:Ascorbic Acid / Q000737:chemistry; D002850:Chromatography, Gel; D003573:Cytochrome b Group / Q000737:chemistry* / Q000302:isolation & purification; D045346:Cytochrome b6f Complex; D019281:Dimerization; D004227:Dithionite / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification; D013056:Spectrophotometry, Ultraviolet; D015204:Spectroscopy, Mossbauer; D018724:Spinacia oleracea / Q000201:enzymology; D013696:Temperature
D003573:Cytochrome b Group; D007506:Iron-Sulfur Proteins; D046911:Macromolecular Substances; D010940:Plant Proteins; C037989:Rieske iron-sulfur protein; D004227:Dithionite; D045346:Cytochrome b6f Complex; D014450:Electron Transport Complex III; D001205:Ascorbic Acid