Electron transfer proteins containing multiple redox centers participate in many metabolically important oxidation-reduction reactions. The function of these proteins depends critically on the arrangement of the redox cofactors and the protein-cofactor interactions that control the redox properties and electron transfer mechanisms. X-ray diffraction methods will be used to determine the three-dimensional structures of five different electron transfer protein systems: (l) the integral membrane protein succinate quinone oxidoreductase (complex II) from Paracoccus denitrificans. (2) the tungstoprotein aldehyde ferredoxin oxidoreductase from the hyperthermophile Pyrococcus furiosus, that contains a tungstopterin analogous to the molybdenum-cofactor. (3) the iron-only hydrogenase from the hyperthermophile ES-4 that contains a structurally uncharacterized iron-sulfur H-cluster in the active site. (4) the copper, thiamin pyrophosphate and iron sulfur containing pyruvate ferredoxin oxidoreductase from P. furiosus. (5) the tungstopterin containing formaldehyde ferredoxin oxidoreductase from the hyperthermophile Thermococcus litoralis. Diffraction quality crystals are available for projects (1) - (3), and will be pursued for (4) - (5). These structures will not only improve understanding of the organization and environment of redox centers in complex electron transfer proteins, but they will also address other significant problems: (i) succinate quinone oxidoreductase represents one of the few integral membrane protein systems to have been crystallized. In addition to the implications for membrane protein folding, analysis of this structure may illuminate factors responsible for the progressive decrease in electron transfer efficiency associated with aging and mitochondrial myopathies. (ii) the aldehyde ferredoxin oxidoreductase and hydrogenase structures will provide structural characterizations of a pterin cofactor and the H- cluster, respectively. (iii) the hyperthermophilic proteins will permit analysis of structural features contributing to extreme thermal stability, which will be central to both an understanding of protein stability and to the design of more stable proteins.
