Alternative Routes for Entry of HgX sub(2) into the Active Site of Mercuric Ion Reductase Depend on the Nature of the X Ligands

• Published in: Biochemistry (Easton) Vol. 38; no. 12; pp. 3519 - 3529
• Main Authors: Engst, S, Miller, S M
• Format: Journal Article
• Language: English
• Published: 01.03.1999
• Subjects: Bacillus; mercury(II) reductase
• ISSN: 0006-2960
• DOI: 10.1021/bi982680c

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) sub(2) substrates. However, CCCC and CCAA enzymes exhibit an equally high rate of binding and turnover using HgBr sub(2) as substrate under pre-steady-state conditions [Engst and Miller (1998) Biochemistry 37, 11496-11507.]. Since the ligands in these HgX sub(2) substrates differ both in size and in affinity for Hg(II), one or both of these properties may contribute to their different reactivities with CCAA enzyme. To further explore the importance of these two properties, we have examined the pre-steady-state reactions of CCCC and CCAA with Hg(CN) sub(2), which has small, high-affinity ligands, and with Hg(Cys) sub(2), which has bulky, high-affinity ligands. The results indicate that HgX sub(2) substrates with small ligands can rapidly access the redox-active cysteines in the absence of the C-terminal cysteines, but those with large ligands require the C-terminal cysteines for rapid access. In addition, it is concluded that the C-terminal cysteines play a critical role in removing the high-affinity ligands before Hg(II) reaches the redox-active cysteines in the inner active site, since direct access of HgX sub(2) substrates with high-affinity ligands leads to formation of an inhibited complex. Consistent with the results, both a narrow channel leading directly to the redoxactive cysteines and a wider 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.