Abstract
Copper-containing nitrite reductases (CuNiRs) play a key role in the global nitrogen cycle by reducing nitrite (NO2−) to nitric oxide, a reaction that involves one electron and two protons. In typical two-domain CuNiRs, the electron is acquired from an external electron-donating partner. The recently characterised Rastonia picketti (RpNiR) system is a three-domain CuNiR, where the cupredoxin domain is tethered to a heme c domain that can function as the electron donor. The nitrite reduction starts with the binding of NO2− to the T2Cu centre, but very little is known about how NO2− binds to native RpNiR. A recent crystallographic study of an RpNiR mutant suggests that NO2− may bind via nitrogen rather than through the bidentate oxygen mode typically observed in two-domain CuNiRs. In this work we have used combined quantum mechanical/molecular mechanical (QM/MM) methods to model the binding mode of NO2− with native RpNiR in order to determine whether the N-bound or O-bound orientation is preferred. Our results indicate that binding via nitrogen or oxygen is possible for the oxidised Cu(II) state of the T2Cu centre, but in the reduced Cu(I) state the N-binding mode is energetically preferred.
Highlights
Microbial copper-containing nitrite reductases (CuNiRs) are enzymes that catalyse the reduction of NO2 − to NO, a key denitrification step in the global nitrogen cycle [1]
Molecular Dynamics of Native and D97N RpNiR in the Resting State
To other CuNiRs, the conserved active site Asp residue, Asp97 in RpNiR is likely a key player for the proton transfer required during catalytic reduction at the type 2 Cu (T2Cu)
Summary
Microbial copper-containing nitrite reductases (CuNiRs) are enzymes that catalyse the reduction of NO2 − to NO, a key denitrification step in the global nitrogen cycle [1]. The most studied and well-characterized are the two-domain CuNiRs, which are homotrimeric proteins with each monomer consisting of two cupredoxin-like domains containing an electron-transfer type 1 Cu (T1Cu) site and a catalytic type 2 Cu (T2Cu) site [2,3]. 12.5 Å and are connected by a Cys-His bridge, with Cys coordination to the T1Cu and His to the T2Cu. Catalytic reduction occurs at the T2Cu site after binding of NO2 − via replacement of the bound water and involves a proton-coupled electron transfer reaction: NO2 − + 2H+ + e− → NO + H2 O. The electron is internally transferred from the T1Cu site to the T2Cu site via the Cys-His bridge and the T1Cu in Molecules 2018, 23, 2997; doi:10.3390/molecules23112997 www.mdpi.com/journal/molecules
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