bCysteine dioxygenases (Cdos), which catalyze the sulfoxidation of cysteine to cysteine sulfinic acid (CSA), have been extensively studied in eukaryotes because of their roles in several diseases. In contrast, only a few prokaryotic enzymes of this type have been investigated. In Ralstonia eutropha H16, two Cdo homologues (CdoA and CdoB) have been identified previously. In vivo studies showed that Escherichia coli cells expressing CdoA could convert 3-mercaptopropionate (3MP) to 3-sulfinopropionate (3SP), whereas no 3SP could be detected in cells expressing CdoB. The objective of this study was to confirm these findings and to study both enzymes in detail by performing an in vitro characterization. The proteins were heterologously expressed and purified to apparent homogeneity by immobilized metal chelate affinity chromatography (IMAC). Subsequent analysis of the enzyme activities revealed striking differences with regard to their substrate ranges and their specificities for the transition metal cofactor, e.g., CdoA catalyzed the sulfoxidation of 3MP to a 3-fold-greater extent than the sulfoxidation of cysteine, whereas CdoB converted only cysteine. Moreover, the dependency of the activities of the Cdos from R. eutropha H16 on the metal cofactor in the active center could be demonstrated. The importance of CdoA for the metabolism of the sulfur compounds 3,3=-thiodipropionic acid (TDP) and 3,3=-dithiodipropionic acid (DTDP) by further converting their degradation product, 3MP, was confirmed. Since 3MP can also function as a precursor for polythioester (PTE) synthesis in R. eutropha H16, deletion of cdoA might enable increased synthesis of PTEs. C ysteine dioxygenases (Cdos) are thiol-oxygenating enzymes that are well characterized in eukaryotes (1, 2). They catalyze the oxidative conversion of cysteine into cysteine sulfinic acid (CSA) and perform the first step in the catabolism of the highly reactive amino acid cysteine (Fig. 1). Because several neurological disorders, like Alzheimer's and Parkinson's diseases (3) and Hallervorden-Spatz disease (4), have been linked to excess levels of cysteine in plasma or the lack of cerebral cysteine dioxygenase activity, the enzyme is exceedingly interesting for medical research.Several analyses of the crystal structure were performed, using recombinant Cdos from different mammalian sources (5-7), and revealed an alternative structural motif for coordination of the iron cofactor by Cdos. Whereas most of the nonheme iron proteins coordinate the metal via two histidine residues and a carboxylic acid group (the 2-His-1-carboxylate facial triad), the ferrous iron in Cdos is arranged in a mutually cis geometry consisting of three histidine residues (3-His facial triad) (1, 8, 9). The loss of Cdo activity after immobilized metal chelate affinity chromatography (IMAC) purification was reported in several studies (10-12), and the activity could be reconstituted only by addition of exogenous ferrous iron, whereas other transition metals failed to restore the activity. In addition, t...