NADH Peroxidase - Reaction Mechanism

Reaction Mechanism

The NADH peroxidase from Enterococcus faecalis is unique in that it utilizes the Cys42 thiol/sulfenic acid (-SH/-SOH) redox couple in the heterolytic cleavage of the peroxide bond to catalyze the two-electron reduction of hydrogen peroxide to water.

The kinetic mechanism of the wild-type peroxidase involves (1) NADH reduction of E(FAD, Cys42-SOH) to EH₂(FAD, Cys42-SH) in an initial priming step; (2) rapid binding of NADH to EH₂; (3) reduction of H₂O₂ by the Cys42-thiolate, yielding E•NADH; and (4) rate-limiting hydride transfer from bound NADH, regenerating EH₂. No discrete FADH₂ intermediate has been observed, however, and the precise details of Cys42-SOH reduction have not been elucidated.

1. E + NADH (EH₂'•NAD+)* EH₂'•NAD+ EH₂ + NAD+ + H₂O
2. EH₂ + NADH EH₂•NADH*
3. EH₂•NADH* + H₂O₂ E•NADH + H₂O
4. E•NADH + H+ EH₂•NAD+ + H₂O
5. EH₂•NAD+ EH₂ + NAD+

Inhibitors include Ag+, Cl-, Co2+, Cu2+, Hg2+, NaN₃, Pb2+, and SO₄2-. At suboptimal H₂O₂ concentrations and concentrations of NADH that are saturating, NADH inhibits the peroxidase activity of the NADH peroxidase by converting the enzyme to an unstable intermediate. NAD+ behaves as an activator by reversing the equilibria that lead to the unstable intermediate, thus converting the enzyme to the kinetically active complex that reduces H₂O₂.