Abstract

Nitrite reductase from Achromobacter xylosoxidans (AxNiR) is a key enzyme in the anaerobic respiratory pathway of denitrification which catalyses the reduction of nitrite (NO2−) to nitric oxide.1 AxNiR belongs to the large family of multi-copper oxidoreductases2 and has been recently shown to have structural similarities to two-domain (small) laccase.3 It is a homo-trimer containing six copper ions found in two different geometries.4, 5 Each monomer contains two distinct copper sites: a buried type 1 (T1) copper site, which receives electrons from external donors, and a type 2 (T2) copper center, located at the monomer–monomer interface, which is the catalytic site where nitrite is bound and reduced to NO (Figure 1). The T1 copper is coordinated by two histidine (His) and a cysteine (Cys) residue in an approximate trigonal planar arrangement, with a relatively weak axial interaction from a methionine (Met) residue (stronger than at most T1 sites). The T2 copper is bound in an approximate tetrahedral arrangement by three His residues, two from one monomer with the third from the adjacent chain, and a water molecule (Figure 1 c). The T1 and T2 copper atoms are 1.24 nm apart in each subunit. The inter-subunit distances between the three T1 Cu centers is 4.35 nm whereas the three T2 Cu centres are 2.96 nm apart. The inter-subunit T1 to T2 copper distances are 3.50 and 3.98 nm (Figure 1 b).

Highlights

  • Nitrite reductase from Achromobacter xylosoxidans (AxNiR) is a key enzyme in the anaerobic respiratory pathway of denitrification which catalyses the reduction of nitrite (NO2À) to nitric oxide.[1]

  • Pulsed electron-electron double resonance (PELDOR)[6] distance measurements provide a tool to structurally characterise this enzyme in solution

  • In this work we have developed a new pulse sequence, inversion-recovery filtered (IRf) PELDOR, which combines the inversion-recovery filter (IRf) technique with PELDOR to eliminate either the type 1 (T1) Cu–Cu or the type 2 (T2) Cu–Cu distances within the multi-copper AxNiR

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Summary

Introduction

Nitrite reductase from Achromobacter xylosoxidans (AxNiR) is a key enzyme in the anaerobic respiratory pathway of denitrification which catalyses the reduction of nitrite (NO2À) to nitric oxide.[1]. Another strategy to separate overlapping EPR signals in biological systems uses differences in relaxation rates.[8] Using pulsed EPR methods, spin–lattice relaxation times (T1 values) of paramagnetic centers can be determined.

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