Dithionite Reduction Kinetics of the Dissimilatory Copper-containing Nitrite Reductase of Alcalegenes xylosoxidans

Abstract

We report here the first detailed study of the dithionite reduction kinetics of a copper-containing dissimilatory nitrite reductase (NiR). The reduction of the blue type 1 copper (T1Cu) center of NiR preparations that contained both type 1 and type 2 copper atoms, followed biphasic kinetics. In contrast, NiR that was deficient in type 2 copper (T2DNiR), followed monophasic kinetics with a second-order rate constant T2D k = 3.06 × 106 m −1s−1. In all cases the SO radical rather thanS2O was the effective reductant. The observed kinetics were compatible with a reaction mechanism in which the T1Cu of the fully loaded protein is reduced both directly by dithionite and indirectly by the type 2 Cu (T2Cu) site via intramolecular electron transfer. Reduction kinetics of the T2Cu were consistent with SO binding first to the T2Cu center and then transferring electrons (112 s−1) to reduce it. As SO is a homologue of NO , the NiR substrate, it is not unlikely that it binds to the catalytic T2Cu site. Effects on the catalytic activity of the enzyme using dithionite as a reducing agent are discussed. Reduction of the semireduced T1Cu(I)T2Cu(II) state followed either second-order kinetics withk 2 = 3.33 × 107 m −1 s −1or first-order kinetics with 52.6 s−1 < 112 s−1. Values of formation constants of the T1Cu(II)T2Cu(II)-SO and T1Cu(I)T2Cu(II)-SO adducts showed that the redox state of T1Cu affected binding of SO at the catalytic T2Cu center. Analysis of the kinetics required the development of a mathematical protocol that could be applied to a system with two intercommunicating sites but only one of which can be monitored. This novel protocol, reported for the first time, is of general application.