E0215

Crystalllographic Study of Electron Transfer Flavoprotein Ubiquinone Oxidoreductase Jian Zhang, David L. Roberts, Frank E. Frerman* and Jung-Ja P. Kim; Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226 . *Department of Pediatrics, University of Colorado School of Medicine, Denver, CO 80262

Electron transfer flavoprotein ubiquinone oxidoreductase (ETF-QO) is a 69kDa membrane-associated protein containing an FAD and an iron-sulfur cluster[1]. ETF-QO and electron transfer flavoprotein (ETF) function as the primary links between acyl-CoA dehydrogenases, which are involved in the (-oxidation of fatty acids as well as degradation of certain amino acids, and the main mitochondrial respiratory chain. Deficiencies in either ETF-QO or ETF result in glutaric acidemia type II, an often fatal human metabolic disease. In an effort to better understand the mechanism of electron transfer through this complex system, we have set out to determine the 3-D structure of ETF-QO by X-ray crystallography. ETF-QO was crystallized in the presence of (-octyl-D-glucopyranoside using the hanging drop vapor diffusion method. The crystals belong to the tetragonal space group P42₁2, with unit cell parameters a=b=154.9A, c=130.3A. Assuming two molecules of ETF-QO per asymmetric unit, the calculated Vm value is 2.8A³/dalton. A native data set has been collected to 3.0A resolution. In an attempt to obtain an isomorphous heavy atom derivative, many different compounds were tried. A single iridium site was located in a crystal soaked in K₂IrCl₆. A second and third derivative were then determined by using cross difference Fourier synthesis from the initial iridium phases. Also, a native anomalous difference Patterson map was used to determine the position of the 4Fe-4S cluster. The overall mean figure of merit was 0.54 for data to 3.2A resolution. Solvent flattening and two-fold non-crystallographic symmetry averaging is presently being used to improve the phases.

Supported by the NIH grant GM29076 (J.-J. P. Kim).

[1] Goodman, S.I., Axtell, K.M., Bindoff, L.A., Beard, S.E., Gill, R.E. and Frerman, F.E. (1994). Eur. J. Biochem. 219:277-286.