In vivo and in vitro effects of mutagenesis of active site tyrosine residues of mercuric reductase.

X-ray crystal structure analysis of mercuric reductase suggested that the binding site for Hg2+ consisted of two tyrosine residues, Tyr264 and Tyr605, as well as two cysteine residues, Cys207 and Cys628. We have previously shown that mutagenesis of Tyr605 to Phe lowered the kcat of the enzyme 6-fold, whereas the same mutation of Tyr264 resulted in a reduction of 160-fold [(1993) Biochemistry 32, 7475-7478]. Tyr605 occupies the same position in mercuric reductase as the active site His residue in the related enzyme glutathione reductase. The mutation of Tyr605 of mercuric reductase to a His residue produced a 24-fold decrease in kcat and a 15-fold decrease in Km. The in vivo resistance to Hg2+ of E. coli strains carrying wild type or mutant merA genes correlated with the in vitro measurements of kcat/Km for mercuric reductase activity.