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Cysteine dioxygenase (CDO) and the flavin reductase, SsuE, are both important proteins in sulfur metabolism. CDO catalyzes cysteine oxidation which is a key step in cysteine metabolism in mammalian systems. SsuE is a flavin reductase found in bacteria that is part of a two-component system involved in sulfur assimilation from alkanesulfonates during sulfur starvation. Following purification of CDO, two bands at ~23 kDa and ~25 kDa were observed by SDS-PAGE. In addition, purified CDO was shown to lose activity over time. A series of experiments were carried out to characterize the two bands and determine what leads to the activity loss of CDO. Results showed that the two bands had the same molecular weight and amino acid sequence, indicating there was no apparent difference between the two proteins. Recent studies from our laboratory have shown that a crosslink between Tyr157 and Cys93 in CDO is responsible for the formation of the lower molecular weight band observed on SDS-PAGE. The participation of the iron center and Tyr157 in the formation of this crosslink may lead to the activity loss of purified CDO protein under aerobic conditions. EPR studies of CDO showed that L-cysteine coordinated to the iron center and altered the environment of the active site. In conclusion, the two bands of CDO have similar molecular weights and amino acid composition, and cysteine binding to the active site alters the environment of the iron center. These studies clarify the composition of these two bands and help to further elucidate the mechanism of CDO. SsuE shares a similar function with the NAD(P)H:flavin reductase (Fre protein) from E. coli that belongs to the ferredoxin-NADP+ (FNR) family. Although there is no significant sequence homology between SsuE and other flavin reductases, a conserved motif, R51XXS54 found in the FNR family is observed in SsuE. Previous studies have shown this motif plays a key role in the recognition of the isoalloxazine ring and flavin catalysis. Based on the role of this motif in the FNR family, the substitution of Ser54 and Phe52 to Ala in SsuE has been performed by site-directed mutagenesis. Kinetic studies of these two mutant proteins showed that Ser54 is involved in flavin binding and Phe52 is likely involved in SsuE catalysis. The pH profile of SsuE suggests that the optimal activity occurs at a pH above 5.0. Future studies will focus on the detailed kinetic analyses of these putative conserved active site residues in SsuE.