CHARACTERIZATION AND CRYSTALLIZATION OF THE LUMEN SIDE DOMAIN OF THE CHLOROPLAST RIESKE IRON-SULFUR PROTEIN

A soluble, 139-residue COOH-terminal polypeptide fragment of the Riesk e iron-sulfur protein of the cytochrome b(6)f complex from spinach chl oroplasts was obtained by Limited proteolysis of the complex and a two -step chromatography purification protocol, The purified Rieske iron-s ulfur protein fragment was characterized by: (i) a single NH2-terminal sequence, NH2-Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of th e intact Rieske protein; (ii) a single molecular weight species determ ined by mass spectrometry with a molecular weight of 14,620 +/- 2 with out the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with re dox- and pH-dependent maxima and minima; and (iv) a reduced-oxidized o ptical difference spectrum characterized by Delta epsilon(mM) = 3.8 mM (-1) cm(-1) for Delta A at 394 versus 409 nm, which was used to determ ine the midpoint oxidation-reduction potential, which is +359 +/- 7 mV at 25 degrees C from pH 5.5-6.5, and +319 +/- 2 mV at pH 7, with an a pparent pK(ox) = 6.5 +/- 0.2 for the oxidized protein The EPR spectrum measured at 17 K was characterized by the g values, g(z) = 2.03 and g (y) = 1.90, and a broad band centered at g(x) approximate to 1.74, ver y similar or identical to those of the Rieske cluster in the b(6)f com plex, implying that the environment of the [2Fe-2S] cluster is similar to that in the complex, Midpoint potential determination by low tempe rature EPR yielded a redox midpoint potential (E(m)) of +365-375 mV of the soluble Rieske fragment at pH 6 and 7 and an E(m) of +295-300 mV of the Rieske cluster in the cytochrome b(6)f complex at pH 6 and 7, T he E(m) difference implies that the environment of the cluster in the soluble Rieske fragment is slightly more polar than that of the cluste r in the intact complex, Single crystals of the Rieske poly-peptide we re obtained that are capable of x-ray diffraction to atomic resolution (<2.5 Angstrom), contain one molecule per assymetric unit, a solvent content of approximately 30%, and belong to the triclinic space group P1 with cell dimensions, a = 29.1 Angstrom b = 31.9 Angstrom, c = 35.8 Angstrom, alpha = 95.6 degrees, beta = 107.1 degrees, gamma = 117.3 d egrees.