Use of a Site-Directed Triple Mutant To Trap Intermediates: Demonstration That the Flavin C(4a)-Thiol Adduct and Reduced Flavin Are Kinetically Competent Intermediates in Mercuric Ion Reductase

A mutant form of mercuric reductase, which has three of its four catalytically essential cysteine residues replaced by alanines (ACAA: Ala₁₃₅Cys₁₄₀Ala₅₅₈Ala₅₅₉), has been constructed and used for mechanistic investigations. With disruption of the Hg(II) binding site, the mutant enzyme is devoid of Hg(II) reductase activity. However, it appears to fold properly since it binds FAD normally and exhibits very tight binding of pyridine nucleotides as is seen with the wild-type enzyme. This mutant enzyme allows quantitative accumulation of two species thought to function as intermediates in the catalytic sequence of the flavoprotein disulfide reductase family of enzymes. NADPH reduces the flavin in this mutant, and a stabilized E-FADH⁻ form accumulates. The second intermediate is a flavin C(4a)-Cys₁₄₀ thiol adduct, which is quantitatively accumulated by reaction of oxidized ACAA enzyme with NADP⁺. The conversion of the Cysi₃₅-Cys₁₄₀ disulfide in wild-type enzyme to the monothiol Cys₁₄₀ in ACAA and the elevated pK_a of Cys₁₄₀ (6.7 vs 5.0 in wild type) have permitted detection of these intermediates at low pH (5.0). The rates of formation of E-FADH⁻ and the breakdown of the flavin C(4a)-thiol adduct have been measured and indicate that both intermediates are kinetically competent for both the reductive half-reaction and turnover by wild-type enzyme. These results validate the general proposal that electrons flow from NADPH to FADH⁻ to C(4a)-thiol adduct to the FAD/dithiol form that accumulates as the EH₂ form in the reductive half-reaction for this class of enzymes.