The nitrite reductase from Pseudomonas aeruginosa: Essential role of two active-site histidines in the catalytic and structural properties

The cytochrome cd1 nitrite reductases are enzymes that catalyse the reduction of nitrite to nitric oxide (NO) in the bacterial energy conversion denitrification process. These enzymes contain two different redox centres: one covalently bound c-haem, which is reduced by external donors, and one peculiar d1-haem, where catalysis occurs. In the present paper, we summarize the current understanding of the reaction of nitrite reduction in the light of the most recent results on the enzyme from Pseudomonas aeruginosa and discuss the differences between enzymes from different organisms. We have evidence that release of NO from the ferrous d1-haem occurs rapidly enough to be fully compatible with the turnover, in contrast with previous hypotheses, and that the substrate nitrite is able to displace NO from the d1-haem iron. These results shed light on the mechanistic details of the activity of cd1 nitrite reductases and on the biological role of the d1-haem, whose presence in this class of enzymes has to date been unexplained.

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Bioinorganic Chemistry of Nickel

Inorganics ◽

10.3390/inorganics7110131 ◽

2019 ◽

Vol 7 (11) ◽

pp. 131

Author(s):

Michael J. Maroney ◽

Stefano Ciurli

Keyword(s):

Active Site ◽

Essential Role ◽

Bioinorganic Chemistry

Following the discovery of the first specific and essential role of nickel in biology in 1975 (the dinuclear active site of the enzyme urease) [...]

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Structure of heme d 1-free cd 1 nitrite reductase NirS

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20006676 ◽

2020 ◽

Vol 76 (6) ◽

pp. 250-256

Author(s):

Thomas Klünemann ◽

Wulf Blankenfeldt

Keyword(s):

Nitric Oxide ◽

Pseudomonas Aeruginosa ◽

Nitrite Reductase ◽

Active Site ◽

Opportunistic Pathogen ◽

Active Enzyme ◽

Free Form ◽

Nitrate Respiration ◽

Gram Negative ◽

Open Conformation

A key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by the cd 1 nitrite reductase NirS in, for example, the Gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d 1 as an essential part of its active site. Although NirS has been well studied mechanistically and structurally, the focus of previous studies has been on the active heme d 1-bound form. The heme d 1-free form of NirS reported here, which represents a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, the movement of a loop around Trp498 seems to be related to a widening of the propeller, allowing easier access to the heme d 1-binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.

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Faculty Opinions recommendation of Essential role of an active-site guanine in glmS ribozyme catalysis.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1095874.550815 ◽

2007 ◽

Author(s):

Robert Batey

Keyword(s):

Active Site ◽

Essential Role ◽

Glms Ribozyme

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The effect of haem ligands on the redox states of the hexa-haem nitrite reductase from Wolinella succinogenes

Biochemical Journal ◽

10.1042/bj2710253 ◽

1990 ◽

Vol 271 (1) ◽

pp. 253-257 ◽

Cited By ~ 4

Author(s):

R S Blackmore ◽

P M A Gadsby ◽

C Greenwood ◽

A J Thomson

Keyword(s):

Ligand Binding ◽

Nitrite Reductase ◽

Active Site ◽

Redox State ◽

Stopped Flow ◽

Ligand Binding Site ◽

Wolinella Succinogenes ◽

Redox States ◽

Cyanide Binding

The nitrite reductase of Wolinella succinogenes containing six covalently bound haem groups has one haem group that will not reduce fully in the presence of excess Na2S2O4. The effect of the extrinsic ligands CO and cyanide on the redox state of this haem was studied by e.p.r. and magnetic c.d. spectroscopy. It was found that both ligands increased the extent of reduction of this haem group, and that in the case of CO binding the level of reduction was correlated with the extent of CO saturation of the enzyme. Stopped-flow studies of the effect of cyanide binding on the rate of dithionite reduction showed that the rate of reduction of the ligand-binding site was increased in the presence of cyanide. This suggests that reduction of the haem groups at the active site is thermodynamically unfavourable in the absence of an extrinsic ligand. The role of the ‘non-reducing’ haem group and the effect of ligands on this centre and on the rate of reduction are discussed in relation to the reduction of nitrite by this enzyme.

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Role of the active-site cysteine of Pseudomonas aeruginosa azurin. Crystal structure analysis of the CuII(Cys112Asp) protein

JBIC Journal of Biological Inorganic Chemistry ◽

10.1007/s007750050157 ◽

1997 ◽

Vol 2 (4) ◽

pp. 464-469 ◽

Cited By ~ 22

Author(s):

Salem Faham ◽

Tadashi J. Mizoguchi ◽

Elinor T. Adman ◽

Harry B. Gray ◽

John H. Richards ◽

...

Keyword(s):

Crystal Structure ◽

Pseudomonas Aeruginosa ◽

Structure Analysis ◽

Active Site ◽

Crystal Structure Analysis ◽

Active Site Cysteine

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Essential Role of an Active-Site Guanine inglmSRibozyme Catalysis

Journal of the American Chemical Society ◽

10.1021/ja0768441 ◽

2007 ◽

Vol 129 (48) ◽

pp. 14858-14859 ◽

Cited By ~ 57

Author(s):

Daniel J. Klein ◽

Michael D. Been ◽

Adrian R. Ferré-D'Amaré

Keyword(s):

Active Site ◽

Essential Role

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Structure of heme d1-free cd1 nitrite reductase NirS

10.1101/2020.02.12.945543 ◽

2020 ◽

Author(s):

Thomas Klünemann ◽

Wulf Blankenfeldt

Keyword(s):

Pseudomonas Aeruginosa ◽

Nitrite Reductase ◽

Active Site ◽

Opportunistic Pathogen ◽

Free Form ◽

Nitrate Respiration ◽

Gram Negative ◽

Open Conformation ◽

Heme D1 ◽

Insight Into

AbstractA key step in anaerobic nitrate respiration is the reduction of nitrite to nitric oxide, which is catalysed by cd1 nitrite reductase NirS in e.g. the gram-negative opportunistic pathogen Pseudomonas aeruginosa. Each subunit of this homodimeric enzyme consists of a cytochrome c domain and an eight-bladed β-propeller that binds the uncommon isobacteriochlorin heme d1 as an essential part of its active site. Although NirS is mechanistically and structurally well studied, the focus of previous studies has been on the active, heme d1-bound form. The heme d1-free form of NirS reported here, representing a premature state of the reductase, adopts an open conformation with the cytochrome c domains moved away from each other with respect to the active enzyme. Further, movement of a loop around W498 seems to be related to a widening of the propeller, allowing easier access to the heme d1 binding side. Finally, a possible link between the open conformation of NirS and flagella formation in P. aeruginosa is discussed.SynopsisThe crystal structure of heme d1-free cd1 nitrite reductase NirS from Pseudomonas aeruginosa has been determined and provides insight into a premature form of the enzyme.

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