Purification and characterization of the Rieske iron-sulfur protein from Thermus thermophilus. Evidence for a [2Fe-2S] cluster having non-cysteine ligands.
We have purified the Rieske iron-sulfur protein from Thermus thermophilus. Chemical analyses show that the protein contains iron, labile sulfide, and cysteine in equimolar concentrations, four of each for Mr approximately 20,000. The oxidized and reduced form of the protein have been characterized by optical, EPR, CD, magnetic CD and Mössbauer spectroscopies. Our data suggest the presence of a unique iron-sulfur center. Mössbauer studies of the oxidized and reduced protein demonstrate unambiguously that the protein contains clusters with [2Fe-2S] cores. The iron analyses and the Mössbauer data, taken together, suggest that the protein has two [2Fe-2S] clusters. This is supported by the observation that two electrons are required for complete reduction of the protein and that the g = 1.94-type signal of the reduced protein has a spin concentration of one spin (S = 1/2) per 2Fe. Within the excellent resolution of the Mössbauer and EPR data, the two clusters are identical. Our results thus suggest that each [2Fe-2S] cluster is coordinated by at most two cysteine residues. The Mössbauer spectra of the reduced protein were analyzed with an S = 1/2 spin Hamiltonian. The hyperfine parameters obtained are very similar to those reported for putidaredoxin. The main difference is that the Rieske protein exhibits an increased isomer shift at the Fe2+ site, suggesting that non-cysteine ligands are coordinated to the site that becomes reduced to Fe2+ upon reduction. A comparison of our data with those reported for various NADH-dependent dioxygenases suggest that these enzymes contain a Rieske-type [2Fe-2S] center.
D000596:Amino Acids / Q000032:analysis; D002942:Circular Dichroism; D003545:Cysteine / Q000032:analysis; D004578:Electron Spin Resonance Spectroscopy; D014450:Electron Transport Complex III; D007501:Iron / Q000032:analysis; D007506:Iron-Sulfur Proteins / Q000302:isolation & purification*; D008667:Metalloproteins / Q000302:isolation & purification*; D008970:Molecular Weight; D013440:Sulfides / Q000032:analysis; D013824:Thermus / Q000032:analysis*
D000596:Amino Acids; D007506:Iron-Sulfur Proteins; D008667:Metalloproteins; C037989:Rieske iron-sulfur protein; D013440:Sulfides; D007501:Iron; D014450:Electron Transport Complex III; D003545:Cysteine