ROLE OF TYROSINE RESIDUES IN HG(II) DETOXIFICATION BY MERCURIC REDUCTASE FROM BACILLUS SP STRAIN RC607

Two tyrosine residues of mercuric reductase (MerA), Tyr-264 and Tyr-605, which were shown by the X-ray crystal structure to be involved in metal binding, were changed to phenylalanine residues by site-directed mutagenesis, both singly (Y264F, Y605F) and to form a double mutant (Y264,605F). The effect of these mutations on Hg(II) reduction activity varied. While MerA Y605F has a similar apparent K(m) to the wild-type enzyme and an apparent k(cat) reduced by 6-fold, MerA Y264F has an apparent K(m) 5-fold lower than the wild type and apparent k(cat) 160-fold lower. The double mutant MerA Y264,605F has the same apparent K(m) as MerA Y264F, but its apparent k(cat) was reduced by a further 7-fold. These results show that the roles of the two tyrosine residues are not equivalent and that Y264 is important for catalysis, possibly by destabilizing the binding of Hg(II) to the two ligating thiolates at the active site of MerA.