Nature's nitrite-to-ammonia expressway, with no stop at dinitrogen

Abstract

Since the characterization of cytochrome c552 as a multiheme nitrite reductase, research on this enzyme has gained major interest. Today, it is known as pentaheme cytochrome c nitrite reductase (NrfA). Part of the NH4+ produced from NO2− is released as NH3 leading to nitrogen loss, similar to denitrification which generates NO, N2O, and N2. NH4+ can also be used for assimilatory purposes, thus NrfA contributes to nitrogen retention. It catalyses the six-electron reduction of NO2− to NH4+, hosting four His/His ligated c-type hemes for electron transfer and one structurally differentiated active site heme. Catalysis occurs at the distal side of a Fe(III) heme c proximally coordinated by lysine of a unique CXXCK motif (Sulfurospirillum deleyianum, Wolinella succinogenes) or, presumably, by the canonical histidine in Campylobacter jejeuni. Replacement of Lys by His in NrfA of W. succinogenes led to a significant loss of enzyme activity. NrfA forms homodimers as shown by high resolution X-ray crystallography, and there exist at least two distinct electron transfer systems to the enzyme. In γ-proteobacteria (Escherichia coli) NrfA is linked to the menaquinol pool in the cytoplasmic membrane through a pentaheme electron carrier (NrfB), in δ- and ε-proteobacteria (S. deleyianum, W. succinogenes), the NrfA dimer interacts with a tetraheme cytochrome c (NrfH). Both form a membrane-associated respiratory complex on the extracellular side of the cytoplasmic membrane to optimize electron transfer efficiency. This minireview traces important steps in understanding the nature of pentaheme cytochrome c nitrite reductases, and discusses their structural and functional features. Graphical abstract

Hydrogen-dependent dissimilatory nitrate reduction to ammonium enables growth of Campylobacterota isolates

“…The significance of the current discovery lies in that the physiological dependency of DNRA sensu stricto on H 2 presence was unequivocally verified in two distinct, genomically characterized isolates. The reaction stoichiometry indicates a tight redox coupling between H 2 oxidation and DNRA, further supported by genome and transcriptome data implicating group 1b [NiFe]-hydrogenases ( hynAB ) and cytochrome c 552 nitrite reductases ( nrfAH ) in this novel pathway (36, 37).…”

Hydrogen-dependent dissimilatory nitrate reduction to ammonium enables growth of Campylobacterota isolates

“…The significance of the current discovery lies in that the physiological dependency of DNRA sensu stricto on H 2 presence was unequivocally verified in two distinct, genomically characterized isolates. The reaction stoichiometry indicates a tight redox coupling between H 2 oxidation and DNRA, further supported by genome and transcriptome data implicating group 1b [NiFe]-hydrogenases ( hynAB ) and cytochrome c 552 nitrite reductases ( nrfAH ) in this novel pathway (36, 37).…”

Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1

“…Kuenenia stuttgartiensis” strain CSTR1 [except for AEC fractions where a low recovery of activity was observed ( Supplementary Figure S9 )]. The nitrite-reducing process was nitrogen balanced ( Figures 1 , 2 ), with no ammonium generated, indicating that no significant activity of dissimilatory nitrite reduction to ammonium (DNRA) was present ( Kroneck, 2022 ). The products of the nitrite-reducing activity were confirmed to be NO, along with N 2 O and N 2 gas.…”

Nitrate–Nitrite Interplay in the Nitrogen Biocycle

“…This occurs through conformational and ligation alterations of hemes c and d 1 , with the latter becoming high spin and ready to bind the substrate. A similar understanding applies to pentaheme cytochrome Nitrite reductase ( cc NiR), where a single high-spin heme defines the reaction site, and to N 2 O reductase, where one of the copper atoms has a lower coordination state [ 6 , 13 , 57 , 58 ].…”

“…The majority of these transformations are catalyzed by microbial enzymes, including nitrogen fixation, nitrification, denitrification, anammox, and dissimilatory nitrate reduction to ammonium (DNRA) [ 4 , 32 ], involving specialized metalloenzymes such as Nitrogenase, [ 11 , 33 ] Nitrate reductases [ 6 , 23 , 30 ], Nitrite oxidoreductase [ 4 , 34 ], and Nitrous oxidoreductase [ 19 , 22 ]. The redox chemistry is associated with redox potentials (standard reduction potentials, E 0 , measured in volts at pH 7) and free energy (ΔG 0 ) [ 35 , 36 ], which play a key role in understanding microbial nitrogen cycling and thermodynamic feasibility [ 11 , 13 , 23 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ].…”

Hydrogen-dependent dissimilatory nitrate reduction to ammonium enables growth of Campylobacterota isolates

“…The significance of the current discovery lies in that the physiological dependency of DNRA sensu stricto on H 2 presence was unequivocally verified in two distinct, genomically characterized isolates. The reaction stoichiometry indicates a tight redox coupling between H 2 oxidation and DNRA, further supported by genome and transcriptome data implicating group 1b [NiFe]-hydrogenases ( hynAB ) and cytochrome c 552 nitrite reductases ( nrfAH ) in this novel pathway (36, 37).…”

Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1

“…Kuenenia stuttgartiensis” strain CSTR1 [except for AEC fractions where a low recovery of activity was observed ( Supplementary Figure S9 )]. The nitrite-reducing process was nitrogen balanced ( Figures 1 , 2 ), with no ammonium generated, indicating that no significant activity of dissimilatory nitrite reduction to ammonium (DNRA) was present ( Kroneck, 2022 ). The products of the nitrite-reducing activity were confirmed to be NO, along with N 2 O and N 2 gas.…”

Nitrate–Nitrite Interplay in the Nitrogen Biocycle

“…This occurs through conformational and ligation alterations of hemes c and d 1 , with the latter becoming high spin and ready to bind the substrate. A similar understanding applies to pentaheme cytochrome Nitrite reductase ( cc NiR), where a single high-spin heme defines the reaction site, and to N 2 O reductase, where one of the copper atoms has a lower coordination state [ 6 , 13 , 57 , 58 ].…”

“…The majority of these transformations are catalyzed by microbial enzymes, including nitrogen fixation, nitrification, denitrification, anammox, and dissimilatory nitrate reduction to ammonium (DNRA) [ 4 , 32 ], involving specialized metalloenzymes such as Nitrogenase, [ 11 , 33 ] Nitrate reductases [ 6 , 23 , 30 ], Nitrite oxidoreductase [ 4 , 34 ], and Nitrous oxidoreductase [ 19 , 22 ]. The redox chemistry is associated with redox potentials (standard reduction potentials, E 0 , measured in volts at pH 7) and free energy (ΔG 0 ) [ 35 , 36 ], which play a key role in understanding microbial nitrogen cycling and thermodynamic feasibility [ 11 , 13 , 23 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ].…”

Hydrogen-dependent dissimilatory nitrate reduction to ammonium enables growth of Campylobacterota isolates

“…The significance of the current discovery lies in that the physiological dependency of DNRA sensu stricto on H 2 presence was unequivocally verified in two distinct, genomically characterized isolates. The reaction stoichiometry indicates a tight redox coupling between H 2 oxidation and DNRA, further supported by genome and transcriptome data implicating group 1b [NiFe]-hydrogenases ( hynAB ) and cytochrome c 552 nitrite reductases ( nrfAH ) in this novel pathway (36, 37).…”

Fractionating proteins with nitrite-reducing activity in “Candidatus Kuenenia stuttgartiensis” strain CSTR1

“…Kuenenia stuttgartiensis” strain CSTR1 [except for AEC fractions where a low recovery of activity was observed ( Supplementary Figure S9 )]. The nitrite-reducing process was nitrogen balanced ( Figures 1 , 2 ), with no ammonium generated, indicating that no significant activity of dissimilatory nitrite reduction to ammonium (DNRA) was present ( Kroneck, 2022 ). The products of the nitrite-reducing activity were confirmed to be NO, along with N 2 O and N 2 gas.…”

Nitrate–Nitrite Interplay in the Nitrogen Biocycle

“…This occurs through conformational and ligation alterations of hemes c and d 1 , with the latter becoming high spin and ready to bind the substrate. A similar understanding applies to pentaheme cytochrome Nitrite reductase ( cc NiR), where a single high-spin heme defines the reaction site, and to N 2 O reductase, where one of the copper atoms has a lower coordination state [ 6 , 13 , 57 , 58 ].…”

“…The majority of these transformations are catalyzed by microbial enzymes, including nitrogen fixation, nitrification, denitrification, anammox, and dissimilatory nitrate reduction to ammonium (DNRA) [ 4 , 32 ], involving specialized metalloenzymes such as Nitrogenase, [ 11 , 33 ] Nitrate reductases [ 6 , 23 , 30 ], Nitrite oxidoreductase [ 4 , 34 ], and Nitrous oxidoreductase [ 19 , 22 ]. The redox chemistry is associated with redox potentials (standard reduction potentials, E 0 , measured in volts at pH 7) and free energy (ΔG 0 ) [ 35 , 36 ], which play a key role in understanding microbial nitrogen cycling and thermodynamic feasibility [ 11 , 13 , 23 , 37 , 38 , 39 , 40 , 41 , 42 , 43 , 44 ].…”