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

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 (T1Cu_N) while the C-terminus contains an extra type 1 Cu (T1Cu_C) bound within a cupredoxin motif. T1Cu_N shows an unusual Cu atom coordination (His₂-Cys-Gln) compared with T1Cu observed in NirKs reported so far (His₂-Cys-Met). T1Cu_C is buried at ∼5 Å from the molecular surface and located ∼14.1 Å away from T1Cu_N; T1Cu_N and T2Cu are ∼12.6 Å apart. All these distances are compatible with an electron-transfer process T1Cu_C → T1Cu_N → T2Cu. T1Cu_N and T2Cu are connected by a typical Cys-His bridge and an unexpected sensing loop which harbours a Ser_CAT 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.

SUBMITTER: Opperman DJ

PROVIDER: S-EPMC6400189 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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

Opperman Diederik Johannes DJ Murgida Daniel Horacio DH Dalosto Sergio Daniel SD Brondino Carlos Dante CD Ferroni Felix Martín FM

IUCrJ 20190209 Pt 2

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 ex ...[more]

PMID: 30867922

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Project description:Herein, we report a three stranded coiled-coil (3SCC) de novo protein containing a type II copper center (CuT2) composed of 6-membered ring N-heterocycles. This design yields the most active homogenous copper nitrite reductase (CuNiR) mimic in water. We achieved this result by controlling three factors. First, previous studies with Nδ and Nε -Methyl Histidine had indicated that a ligand providing pyridine-like electronic character to the copper site was superior to the more donating Nδ for nitrite reduction. By substitution of the parent histidine with the non-coded amino acids pyridyl alanine (3'-Pyridine [3'Py] vs 4'-Pyridine [4'Py]), an authentic pyridine donor was employed without the complications of the coupling of both electronic and tautomeric effects of histidine or methylated histidine. Second, by changing the position of the nitrogen atom within the active site (4'-Pyridine vs. 3'Pyridine) a doubling of the enzyme's catalytic efficiency resulted. This effect was driven exclusivity by substrate binding to the copper site. Third, we replaced the leucine layer adjacent to the active site with an alanine, and the disparity between the 3'Py and 4'Py became more apparent. The decreased steric bulk minimally impacted the 3'Py derivative; however, the 4'Py K m decreased by an order of magnitude (600 mM to 50 mM), resulting in a 40-fold enhancement in the k cat/K m compared to the analogues histidine site and a 1500-fold improvement compared with the initially reported CuNiR catalyst of this family, TRIW-H. When combined with XANES/EXAFS data, the relaxing of the Cu(I) site to a more 2-coordinate Cu(I) like structure in the resting state increases the overall catalytic efficiency of nitrite reduction via the lowering of K m. This study illustrates how by combining advanced spectroscopic methods, detailed kinetic analysis, and a broad toolbox of amino acid side chain functionality, one can rationally design systems that optimize biomimetic catalysis.

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

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

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