Flavoproteins in Oxidative Protein Folding

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DESCRIPTION (provided by applicant): Disulfide bonds critical to the native structure of secreted proteins, and to the regulation of a range of biochemical processes, however their biosynthesis in multicellular organisms remains poorly understood. This proposal continues investigation of a newly-recognized flavoenzyme family (the Quiescin-sulfhydryl oxidase, QSOX, family) formed from the fusion of two thioredoxin domains with a 4-helix bundle FAD-binding domain. Thioredoxin and flavin-binding domains cooperate to ensure the facile insertion of disulfide bonds into unfolded reduced proteins with the reduction of oxygen to hydrogen peroxide. Three aims of this proposal use the non-recombinant avian sulfhydryl oxidase as the prototypical member of this new flavoenzyme family. The cooperation between protein disulfide isomerase (PDI) and QSOX in oxidative folding will be investigated by steady-state and rapid-reaction kinetics. The existence of a binding site on QSOX for peptides and unfolded reduced proteins will be assessed by kinetic and spectroscopic experiments. The inhibition of QSOX during turnover in the presence of arsenic(Ill) compounds or transition metals will be examined for insights into ways to modulate sulfhydryl oxidase activity in vivo. The fourth aim, heterologous expression of both avian and human QSOX enzymes, will permit a mutational dissection of the roles of the three CxxC motifs in catalysis. Expression of a human QSOX (QSOX- 1) will allow the first detailed characterization of its catalytic specificity towards small and large thiol substrates. The fifth and sixth aims extend this work to the enzyme's smaller, stand-alone, flavoprotein cousins: human recombinant ALR (augmenter of liver regeneration) and Erv2p (the closest functional counterpart of QSOX in yeast).