The function of the metalloenzymes is mainly determined by four structural features: the metal core, the metal binding motif, the second sphere residues in the active site and the electronic statistics. Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are the only known enzymes that oxidize free thiol containing molecules in mammals by inserting of a dioxygen molecue. Both ADO and CDO are known as non-heme iron dependent enzymes with 3-His metal binding motif. However, the mechanistic understanding of both enzymes is obscure. The understanding of the mechanistic features of the two thiol dioxygenases is approached through spectroscopic and metal substitution in this dissertation. Another focus of the dissertation is the understanding of the function of a second sphere residue His228 in a 3-His-1-carboxyl zinc binding decarboxylase α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD). ACMSD catalyzes the decarboxylation through a hydrolase-like mechanism that is initialized by the deprotonation of metal bounded water molecule. Our study reveled that the second sphere residue His228 is responsible for the water deprotonation through hydrogen bonding. The spectroscopic and crystallographic data showed the H228Y mutation binds ferric iron instead of native zinc metal and the active site water is replaced by the Tyr228 residue ligation. Thus, we concluded that, H228Y not only plays a role of stabilizing and deprotonating the active site water but also is an essential residue on metal selectivity.