A three-domain copper-nitrite reductase with a unique sensing loop.

Diederik Johannes Opperman,Daniel Horacio Murgida,Carlos Dante Brondino,Sergio Daniel Dalosto,Felix Martín Ferroni

doi:10.1107/s2052252519000241

Abstract

Dissimilatory nitrite reductases are key enzymes in the denitrification pathway, reducing nitrite and leading to the production of gaseous products (NO, N2O and N2). The reaction is catalysed either by a Cu-containing nitrite reductase (NirK) or by a cytochrome cd 1 nitrite reductase (NirS), as the simultaneous presence of the two enzymes has never been detected in the same microorganism. The thermophilic bacterium Thermus scotoductus SA-01 is an exception to this rule, harbouring both genes within a denitrification cluster, which encodes for an atypical NirK. The crystal structure of TsNirK has been determined at 1.63 Å resolution. TsNirK is a homotrimer with subunits of 451 residues that contain three copper atoms each. The N-terminal region possesses a type 2 Cu (T2Cu) and a type 1 Cu (T1CuN) while the C-terminus contains an extra type 1 Cu (T1CuC) bound within a cupredoxin motif. T1CuN shows an unusual Cu atom coordination (His2-Cys-Gln) compared with T1Cu observed in NirKs reported so far (His2-Cys-Met). T1CuC is buried at ∼5 Å from the molecular surface and located ∼14.1 Å away from T1CuN; T1CuN and T2Cu are ∼12.6 Å apart. All these distances are compatible with an electron-transfer process T1CuC → T1CuN → T2Cu. T1CuN and T2Cu are connected by a typical Cys-His bridge and an unexpected sensing loop which harbours a SerCAT residue close to T2Cu, suggesting an alternative nitrite-reduction mechanism in these enzymes. Biophysicochemical and functional features of TsNirK are discussed on the basis of X-ray crystallography, electron paramagnetic resonance, resonance Raman and kinetic experiments.

Highlights

The global nitrogen cycle maintained by some bacteria impacts all forms of life worldwide (Zumft, 1997; Gruber & Galloway, 2008; Fowler et al, 2014)
The overall structure of TsNirK [Fig. 2(a)] shows a unique distribution of domains and subunit interactions that differs greatly from those reported for Hyphomicrobium denitrificans A3151 (HdNir) (PDB entry 2dv6; Nojiri et al, 2007), RpNir (PDB entry 3ziy; Antonyuk et al, 2013) and PhNir (PDB entry 2zoo; Tsuda et al, 2013) [Fig. 1(c)]
HdNir, RpNir and PhNir have an extra C-terminal or N-terminal domain harbouring a haem c or a T1Cu cofactor that does not interact with the two-domain core of the same subunit (Antonyuk et al, 2013; Nojiri et al, 2007; Tsuda et al, 2013) [Fig. 1(c)]

Summary

Introduction

The global nitrogen cycle maintained by some bacteria impacts all forms of life worldwide (Zumft, 1997; Gruber & Galloway, 2008; Fowler et al, 2014). The biological fixation of atmospheric dinitrogen to produce NH3 is the process that introduces inorganic nitrogen into the biosphere, while the denitrification process proceeds in the opposite direction. Bacteria convert inorganic nitrogen into organic nitrogen sources by assimilatory pathways during the interconversion of NH3, NOÀ3 and NOÀ2. Dissimilatory denitrification produces dinitrogen by the reduction of NOÀ3 and NOÀ2 , with NO and N2O as intermediaries involving several enzymes in the process. NO + H2O), catalysed by nitrite reductase (Nir), is the key reaction that initiates the dissimilatory denitrification process in denitrifiers (Zumft, 1997). Two kinds of Nirs are involved in this catalytic step, the haem- and copper-containing enzymes, NirS and

Methods

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Conclusion