A QM/MM Study of Nitrite Binding Modes in a Three-Domain Heme-Cu Nitrite Reductase

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.

QM/MM Simulations of Protein Crystal Reactivity Guided by MSOX Crystallography: A Copper Nitrite Reductase Case Study

“…Figure shows the MSOX and calculated structures of NO 2 – bound in the bidentate top-hat orientation to oxidized Cu­(II) at the T2Cu site. Further analysis of the geometries according to the method of ref for different protonation states of Asp CAT and His CAT is provided in the SI, Table S1.…”

“…One possibility is that the orientation is sensitive to subtle differences between the SF-ROX and MSOX structures. For comparison in a three-domain CuNiR Asp CAT to Asn mutant, Rp NiR, an L-shaped N-bound nitrite orientation that resembles a distorted side-on orientation was obtained with the Cu­(II) state, , and ordinary dynamicity in the active site could modulate the nitrite orientation too. Another possibility is that the protonation states of other residues besides Asp CAT and His CAT are important in determining the nitrite orientation, although it is not obvious which other residues may be responsible.…”

Multiscale QM/MM modelling of catalytic systems with ChemShell

“…104 Wu et al used QM/MM calculations in ChemShell to investigate the form of the active species in two binuclear copper monooxygenases, which catalyse stereospecific C–H hydroxylation of precursors to hormones and neurotransmitters, resulting in a full description of the catalytic cycle. 105 Sen et al have investigated copper-containing nitrite reductases, 106 which reduce nitrite to nitric acid as part of the global nitrogen cycle. These enzymes contain two copper centres, one of which catalyses the reduction reaction while the other is involved in electron transfer.…”