Flavoproteins constitute one of the largest groups of related proteins presently pathway including those involved in biosynthesis, biodegradation, and energy transduction in such diverse and essential life processes as oxidative-phosphorylation, photosynthesis, and nitrogen fixation. The number and variety of reactions involving flavoproteins attests to the remarkable versatility of the common flavin cofactor and suggests that the apoprotein exerts strict control over the redox and chemical properties of the bound flavin. Two overall objectives of our research are: 1) to learn how the specific interactions between apoprotein and flavin are involved in the control of the redox properties of the cofactor, and 2) to investigate how specific amino acid residues in the vicinity of the flavin cofactor influence and regulate electron transfer from the flavin to redox centers in other redox proteins. In this proposal we describe the continuation of a new approach to this fundamental problem in biology and life sciences. We have established a genetic system with which to apply recombinant DNA technology to engineer specific amino acid substitutions in the flavin binding site of the simplest and best characterized class of flavoprotein, the flavodoxin. The detailed X-ray crystal structures of four different flavodoxins have revealed, at the atomic level, specific features of flavin-protein interactions. Through protein engineering, we are able to determine which of these interaction are functionally critical and in what way. Fundamental properties under investigation include the unusually low redox potential of the flavin hydroquinone and the stabilization of the blue neutral semiquinone which results in the separation of these two redox couples in a way that is physiologically important as well as the structural features essential in electron transfer between the flavodoxin and donor/acceptor redox proteins such as certain cytochromes. This proposal describes a multifaceted approach in the generation of families of related flavodoxin proteins differing only in specific and well-defined regions of the flavin binding site and the application of a variety of spectroscopic approaches to assist us in establishing a correlation between these altered structural features and observed changes in the redox properties of the bound flavin cofactor.
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