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 Rieske iron-sulfur protein of the cytochrome b(6)f complex from spinach chloroplasts was obtained by Limited proteolysis of the complex and a two-step chromatography purification protocol, The purified Rieske iron-sulfur protein fragment was characterized by: (i) a single NH2-terminal sequence, NH2-Phe-Val-Pro-Pro-Gly-Gly, starting with residue 41 of the intact Rieske protein; (ii) a single molecular weight species determined by mass spectrometry with a molecular weight of 14,620 +/- 2 without the [2Fe-2S] cluster; (iii) an optical absorbance spectrum with redox- and pH-dependent maxima and minima; and (iv) a reduced-oxidized optical 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 determine 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 apparent 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, very similar or identical to those of the Rieske cluster in the b(6)f complex, implying that the environment of the [2Fe-2S] cluster is similar to that in the complex, Midpoint potential determination by low temperature 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, The E(m) difference implies that the environment of the cluster in the soluble Rieske fragment is slightly more polar than that of the cluster in the intact complex, Single crystals of the Rieske poly-peptide were 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 degrees.