Alternative routes for entry of HgX2 into the active site of mercuric ion reductase depend on the nature of the X ligands

Wild-type mercuric ion reductase (CCCC enzyme) possesses four cysteines in each of its Hg(II) binding sites, a redox-active pair and a C-terminal pair . Mutation of the C-terminal cysteines to alanines (CCAA enzyme) leads to a loss of steady-state mercuric ion reductase activity using Hg(SR)(2) subst rates. However, CCCC and CCAA enzymes exhibit an equally high rate of bindi ng and turnover using HgBr2 as substrate under pre-steady-state conditions [Engst and Miller (1998) Biochemistry 37, 11496-11507.]. Since the ligands in these HgX2 substrates differ both in size and in affinity for Hg(II), on e or both of these properties may contribute to their different reactivitie s with CCAA enzyme. To further explore the importance of these two properti es, we have examined the pre-steady-state reactions of CCCC and CCAA with H g(CN)(2), which has small, high-affinity ligands, and with Hg(Cys)(2), whic h has bulky, high-affinity ligands. The results indicate that HgX2 substrat es with small ligands can rapidly access the redox-active cysteines in the absence of the C-terminal cysteines, but those with large ligands require t he C-terminal cysteines for rapid access. In addition, it is concluded that the C-terminal cysteines play a critical role in removing the high-affinit y ligands before Hg(II) reaches the redox-active cysteines in the inner act ive site, since direct access of HgX2 substrates with high-affinity ligands leads to formation of an inhibited complex. Consistent with the results, b oth a narrow channel leading directly to the redox-active cysteines and a w ider channel leading to the redox-active cysteines via initial contact with the C-terminal cysteines can be identified in the structure of the enzyme from Bacillus sp. RC607.