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AbstractFlavin-linked sulfhydryl oxidases participate in the net generation of disulfide bonds during oxidative protein folding in the endoplasmic reticulum. Members of the Quiescin-sulfhydryl oxidase (QSOX) family catalyze the facile direct introduction of disulfide bonds into unfolded reduced proteins with the reduction of molecular oxygen to generate hydrogen peroxide. Current progress in dissecting the mechanism of QSOX enzymes is reviewed, with emphasis on the CxxC motifs in the thioredoxin and Erv/ALR domains and the involvement of the flavin prosthetic group. The tissue distribution and intra- and extracellular location of QSOX enzymes are discussed, and suggestions for the physiological role of these enzymes are presented. The review compares the substrate specificity and catalytic efficiency of the QSOX enzymes with members of the Ero1 family of flavin-dependent sulfhydryl oxidases: enzymes believed to play key roles in disulfide generation in yeast and higher eukaryotes. Finally, limitations of our current understanding of disulfide generation in metazoans are identified and questions posed for the future. Antioxid. Redox Signal.13, 1217–1230. [ABSTRACT FROM AUTHOR]

The su1fhdryI oxidase augmenter of liver regeneration (ALR) binds FAD in a helix-rich domain that presents a CxxC disulfide proximal to the isoalloxazine ring of the flavin. Head-to-tail interchain disulfide bonds link subunits within the homodimer of both the short. cytokine-like. form of ALR (5fALR), and a longer form (IfALR) which resides in the mitochondrial interniembrane space (IMS). IfALR has an 80-residue N-terminal extension with an additional CxxC motif required for the reoxidation of reduced Mia4O during oxidative protein folding within the IMS. Recently, Di Fonzo et al. [Di Fonzo, A., Ronchi, D., Lodi. T., Fassone, E., Tigano, M., Lamperti, C., Corti, S., Bordoni. A., Fortunato, F., Nizzardo, M., Napoli. L., Donadoni, C., Salani, S., Saladino, F., Moggio, M., Bresolin, N., Ferrero, I.. and Comi, G. p. (2009) Am. J. Hu,n. Gene!. 84, 594-6041 described an R194H mutation of human ALR that led to cataract, progressive muscle hypotonia, and hearing loss in three children. The current work presents a structural and enzymological characterization of the human RI94H mutant in If- and sfALR. A crystal structure of human sfALR was determined by molecular replacement using the rat sfALR structure. R194 is located at the subunit interface of sfALR. close to the intersubunit disulfide bridges. The R194 guanidino moiety participates in three H-bonds: two main-chain carbonyl oxygen atoms (from R194 itself and from C95 of the intersubunit disulfide of the other protomer) and with the 2'-OH of the FAD ribose. The R194H mutation has minimal effect on the enzyme activity using model and physiological substrates of short and long ALR forms. However, the mutation adversely affects the stability of both ALR forms: e.g., by decreasing the melting temperature by about 10°C, by increasing the rate of dissociation of FAD from the holoenzyme by about 45-fold, and by strongly enhancing the susceptibility of sfALR to partial proteolysis and to reduction of its intersubunit disulfide bridges by glutathione. Finally, a comparison of the TROSY-HSQC 2D NM R spectra of wild-type sfALR and its R194H mutant reveals a significant increase in conformational flexibility in the mutant protein. In sum, these in vitro data document the major impact of the seemingly conservative R194H mutation on the stability of dimeric ALR and complement the in vivo observations of Di Fonzo et al. [ABSTRACT FROM AUTHOR]