E COLI CYTOCHROME BD OXIDASE

这个项目的主要目的是获得一个连贯的结构和

This project aims mainly at obtaining a coherent view of the structure and functional mechanism of the oxygen-reducing domain of the cytochrome bd ubiquinol oxidase from Escherichia coil. This enzyme is the only well characterized respiratory oxidase which does not belong to the family of heme-copper oxidases. All members of the heme-copper oxidase family, which includes the eukaryotic cytochrome c oxidase, have in common a bimetallic center, consisting of a heme iron and a copper atom, which is where molecular oxygen is reduced to water. Cytochrome bd, in contrast, contains no copper. This enzyme contains a total of three heme prosthetic groups: heme b558, heme b595, and heme d. Heme b558 appears to be simply involved in the transfer of electrons to the site where oxygen is reduced Past work from numerous laboratories has shown that heme d is five-coordinate and binds to oxygen directly. However, the role of heme b595 is not known. Heme b595 is also five-coordinate and, thus, in principle, Could also bind to exogenous ligands, including oxygen. Much of the proposed work is directed towards defining the ligand binding properties of both heme b595 and heme d. Spectroscopic methods, including magnetic circular dichroism (MCD) and circular dichroism (CD) will be used to characterize both the complexes that form and the kinetics of their formation under a variety of environmental conditions. Preliminary data have demonstrated the ability of these techniques to define the coordination and spin-state of each of the hemes in the presence of exogenous ligands. A variety of different ligands will be used to probe both the reduced and oxidized forms of the oxidase, including cyanide, azide, CO, and fluoride as well as others. Oxygenated forms of the enzyme will be generated by reacting the oxidase with peroxides and with molecular oxygen. The dependence of these reactions on the pH of the periplasmic and of the cytoplasmic sides of the membrane will be examined in reconstituted proteoliposomes using established procedures. By using these approaches, the role of heme b595 and any cooperative function of this heme with heme d should become clear. Quite possibly, heme b595 and heme d form a bimetallic center analogous to the well known heme-copper center found in most other oxidases. An active collaboration with Dr. Konstantinov, our foreign collaborator, has already been established over the past two years, and preliminary results have demonstrated both the utility of this approach and that these studies can be performed in his laboratory at Moscow State University.