Recombinant Pseudomonas aeruginosa Protein nirH (nirH)
Description
Genetic Organization and Functional Context
NirH is part of the nir gene cluster (nirSMCFDLGHJEN), which encodes proteins required for heme d₁ biosynthesis and nitrite reduction . The cluster includes:
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NirS: Catalyzes nitrite reduction to nitric oxide (NO).
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NirM, NirC, NirF: Involved in heme d₁ precursor formation.
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NirD, NirL, NirG, NirH: Compose the siroheme decarboxylase complex, converting siroheme to 12,18-didecarboxy-siroheme, an intermediate in heme d₁ biosynthesis .
Table 1: nir Gene Cluster Components and Their Roles
| Gene | Protein Function | Role in Heme d₁ Biosynthesis |
|---|---|---|
| nirS | Nitrite reductase | Reduces nitrite to NO |
| nirD, nirL, nirG, nirH | Siroheme decarboxylase | Removes carboxyl groups from siroheme |
| nirE | S-Adenosylmethionine-dependent methyltransferase | Methylates uroporphyrinogen III |
| nirN | Heme d₁ synthase | Introduces acrylic double bond to dihydro-heme d₁ |
| nirF | Unknown | Required for heme d₁ maturation |
Sequence Homology
NirH shares sequence homology with NirD (68.5% identity), NirL (60.4%), and NirG (63.9%) . These proteins form a decarboxylase complex that removes carboxyl groups from siroheme, a precursor of heme d₁.
Mutational Analysis
Deletion of nirH (strain RM361) results in a complete loss of nitrite reductase (NirS) activity, as demonstrated by enzyme assays and heme staining . This confirms NirH’s indispensable role in heme d₁ biosynthesis.
Table 2: Impact of nirH Mutation on NirS Activity
| Strain (Genotype) | NirS Activity (mU/mg protein) | Heme d₁ Presence |
|---|---|---|
| PAO1 (wild type) | 48.3 | Yes |
| RM361 (nirH::tet) | Not Detected (ND) | No |
Heme d₁ Biosynthesis
The pathway involves:
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Uroporphyrinogen III methylation by NirE to form precorrin-2 .
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Decarboxylation by NirD, NirL, NirG, and NirH to yield 12,18-didecarboxy-siroheme .
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Final maturation by NirN, introducing a double bond to form heme d₁ .
Functional Interdependence
NirH’s activity is tightly linked to NirD, NirL, and NirG. Mutations in any of these genes (e.g., nirD, nirL, nirG) also abolish NirS activity, highlighting the cooperative nature of the complex .
Denitrification and Pathogenicity
Denitrification allows P. aeruginosa to thrive in anaerobic environments, such as biofilms or host tissues. NirH’s role in this pathway underscores its potential as a therapeutic target to disrupt bacterial survival in infections .
Biotechnological Applications
Recombinant NirH could be used to study heme d₁ biosynthesis in vitro or engineer denitrification pathways in industrial microbes for bioremediation .
Product Specs
Target Background
KEGG: pae:PA0512
STRING: 208964.PA0512
Q&A
What is the enzymatic role of NirH in Pseudomonas aeruginosa denitrification, and how does it influence experimental design?
NirH is a critical component of the siroheme decarboxylase complex (NirD/NirL/NirG/NirH) responsible for converting siroheme to 12,18-didecarboxy-siroheme during heme d1 biosynthesis . This cofactor is essential for the nitrite reductase NirS, which drives denitrification.
Methodological considerations:
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Pair UV-visible absorption spectroscopy and high-resolution mass spectrometry to confirm cofactor intermediates (e.g., dihydro-heme d1) .
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Include 5-aminolevulinic acid in growth media to enhance heme precursor availability .
How do researchers validate NirH interaction partners in protein networks?
NirH participates in multi-protein complexes with denitrification enzymes. Key interaction data from affinity purification studies :
Advanced validation methods:
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Combine co-purification assays with in vivo immunogold labeling to confirm spatial colocalization .
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Use norB or nirS mutants to assess compensatory interactions in the protein network .
What strategies resolve contradictions in NirH cofactor binding affinity data?
Discrepancies often arise from oxygen sensitivity or incomplete cofactor synthesis:
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Anaerobic purification: Perform protein isolation in an oxygen-free chamber to preserve labile intermediates .
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Cofactor reconstitution: Incubate apo-NirH with synthetic dihydro-heme d1 under reducing conditions .
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Cross-validation: Compare resonance Raman spectroscopy (structural insights) with enzymatic activity assays (functional confirmation) .
Which structural analysis methods are suitable for NirH given its metalloprotein complexity?
Advanced approaches:
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Cryo-EM: Resolve large NirH-containing complexes (e.g., siroheme decarboxylase) at 3–4 Å resolution .
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EPR spectroscopy: Characterize iron-sulfur clusters in NirH under varying redox states .
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Molecular dynamics simulations: Model electron transfer pathways between NirH and NorB/NarH .
