STRUCTURES OF ELECTRON TRANSFER FLAVOPROTEIN FROM HUMAN AND PARACOCCUS DENITRIFICANS. David L. Roberts, Frank E. Frerman^[[section]], and Jung-Ja P. Kim, Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI 53226, ^[[section]]University of Colorado Health Science Center, Denver Colorado, 80262.

Mammalian electron transfer flavoprotein (ETF) contains a single equivalent of FAD per aß dimer, and functions as an electron shuttle between the primary dehydrogenases that are involved in fatty acid and amino acid metabolism and the membrane bound electron transfer flavoprotein dehydrogenase (ETF-QO). ETF from Paracoccus denitrificans has been shown to be highly homologous with the human enzyme, showing > 55% amino acid sequence identity.

The structure of the human ETF protein has been solved to 2.1 Å. The enzyme crystallizes in the monoclinic space group P2₁, with unit cell parameters a=47.46, b=104.92, c=63.79 Å, and ß=110.09deg.. The phases were solved using MIR, with three heavy atom derivatives. Density modification procedures including solvent flattening and phase combination with SigmaA were used to improve the initial phases. The refinement was carried out using XPLOR, with alternating rounds of manual adjustment of the model. The final R factor without added waters is 23.3%, with R_free=30.3% for all reflections between 15-2.1 Å.

Using this refined human ETF model, molecular replacement was used to solve the P. denitrificans ETF structure to 2.5Å. The P. denitrificans ETF crystallizes in the orthorhombic space group P2₁2₁2₁, with unit cell parameters a=70.52, b=80.13, and c=184.00 Å. After initial refinement, the R-factor is 27.5%, with R_free=38.5%. We are presently refining the model.

This work was supported by NRSA DK09157-02 (DLR) and NIH grant GM29076 (JJPK).