A series of mutations was targeted at the methionine residue, Met471, coordinating the Cu-M site of tyramine beta-monooxygenase (T beta M). The methionine ligand at Cu-M is believed to be key to dioxygen activation and the hydroxylation chemistry of the copper monooxygenases. The reactivity and copper binding properties of three T beta M mutants, Met471Asp, Met471Cys, and Met471His, were examined. All three mutants show similar metal binding affinities to wild type T beta M in the oxidized enzyme forms. EPR spectroscopy suggests that the Cu-II coordination geometry is identical to that of the WT enzyme. However, substrate hydroxylation was observed for the reaction of tyramine solely with Met471Cys T beta M. Met471Cys T beta M provides the first example of an active mutant directed at the Cum site of this class of hydroxylases. The reactivity and altered kinetics of the Met471Cys mutant further highlight the central role of the methionine residue in the enzyme mechanism. The sole ability of the cysteine residue to support activity among the series of alternate amino acids investigated is relevant to theoretical and biomimetic investigations of dioxygen activation at mononuclear copper centers.