Direct Electrochemistry of Nitrite Reductase from Achromobacter cycloclastes IAM 1013

The cyclic voltammetry of the nitrite reductase from Achromobacter cycloclastes IAM 1013 exhibited well defined voltammetric response at a di-4-pyridyl disulfide (4-pyds) modified gold electrode in the presence of A. cycloclastes apopseudoazurin. The midpoint potential, E1/2, of the native and type II copper-depleted (T2D) nitrite reductase obtained from the voltammogram were estimated to be 240 mV and 204 mV vs. NHE, respectively. The almost identical current value of the native and T2D reductase suggested that the voltammetric behavior contributed from the type 1 copper site. When nitrite was added into the nitrite reductase solution (pH 7.0), an enhanced sigmoidal cathodic current-potential curve indicating the catalytic regeneration of oxidized nitrite reductase was observed. The rate constant of nitrite reduction and the Michaelis constant, Km, of the native reductase were estimated to be 5 × 102 mol−1 dm3 s−1 and 7 × 10−4 mol dm−3, respectively. The rate constant of the nitrite reduction and the Km value of T2D nitrite reductase were also estimated to be 1 × 102 mol−1 dm3 s−1 and 3 × 10−3 mol dm−3, respectively. These results suggested that the nitrite reduction activity is closely related with type II copper site.