Recombinant Nitrosomonas europaea 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (ispH)

What is the structure of ispH and how does it relate to its function?

IspH contains a catalytically essential [4Fe-4S] cluster in its active site, which undergoes redox changes during the reaction cycle . The enzyme exists in multiple redox states during catalysis:

• Resting state: Contains a diamagnetic [4Fe-4S]2+ cluster

• Reduced state: Upon reduction, generates [4Fe-4S]+ with both S = 1/2 and S = 3/2 spin ground states

• Intermediate state: When the reduced enzyme interacts with substrate, forms a transient paramagnetic reaction intermediate thought to contain a cluster-bound substrate-derived species

• Product-bound state: Incubation with product (IPP) induces another paramagnetic [4Fe-4S]+ species with S = 1/2

This redox cycling of the [4Fe-4S] cluster is critical for ispH's ability to catalyze the reductive dehydroxylation of HMBPP. The iron-sulfur cluster serves as both an electron transfer center and a substrate binding site, with the substrate likely coordinating directly to an iron atom of the cluster during catalysis.

How does recombinant expression affect ispH properties compared to native expression in Nitrosomonas europaea?

Recombinant expression of ispH from Nitrosomonas europaea presents both advantages and challenges compared to studying the native enzyme. While recombinant systems allow for higher protein yields and easier purification through affinity tags, they may introduce subtle differences in enzyme properties.

• Iron-sulfur cluster assembly machinery efficiency

• Post-translational modifications

• Protein folding kinetics and chaperone availability

• Redox environment

These differences could potentially impact the catalytic properties, stability, and spectroscopic features of the recombinant enzyme. Researchers should validate recombinant ispH by comparing its spectroscopic signatures (particularly EPR and Mössbauer spectra) and kinetic parameters with those expected for properly folded and active enzyme.

What spectroscopic techniques are most informative for investigating the reaction mechanism of recombinant Nitrosomonas europaea ispH?

A multifaceted spectroscopic approach provides the most comprehensive insights into ispH's reaction mechanism:

• Electron Paramagnetic Resonance (EPR): Detects paramagnetic species formed during the catalytic cycle, allowing characterization of the S = 1/2 and S = 3/2 spin states of the reduced [4Fe-4S]+ cluster and transient reaction intermediates .

• 57Fe Electron-Nuclear Double Resonance (ENDOR): When using 57Fe-labeled enzyme preparations, ENDOR enables determination of hyperfine tensors for all four iron atoms in the [4Fe-4S] cluster, providing detailed information about electron delocalization and substrate interactions .

• Mössbauer Spectroscopy: With 57Fe-labeled samples, Mössbauer spectroscopy confirms cluster oxidation states and detects subtle changes in iron environments during catalysis .

• UV-Visible Absorption Spectroscopy: Monitors changes in the [4Fe-4S] cluster environment during substrate binding and turnover.

• Fourier-Transform Infrared (FTIR) Spectroscopy: Provides information about substrate binding orientation and changes in protein secondary structure.

These complementary techniques allow researchers to build a comprehensive model of the electron transfer events, substrate interactions, and conformational changes occurring during the ispH reaction cycle.

What are the challenges in maintaining [4Fe-4S] cluster integrity during recombinant expression and purification of Nitrosomonas europaea ispH?

Maintaining [4Fe-4S] cluster integrity during recombinant expression and purification presents several significant challenges:

• Oxygen sensitivity: [4Fe-4S] clusters are highly susceptible to oxidative damage, necessitating strictly anaerobic conditions throughout handling.

• Iron and sulfur incorporation: Efficient in vivo assembly of [4Fe-4S] clusters requires adequate iron and sulfur availability in the expression host. Supplementation with iron sources (such as ferric ammonium citrate) may be necessary.

• Expression system selection: The choice of expression host affects iron-sulfur cluster assembly efficiency. E. coli strains with robust iron-sulfur cluster assembly machinery are preferred.

• Buffer composition: Purification buffers must contain reducing agents (DTT or β-mercaptoethanol) to prevent oxidative damage, along with glycerol as a stabilizing agent.

• Reconstitution requirements: Partial loss of [4Fe-4S] clusters often occurs during purification, requiring in vitro reconstitution using chemical or enzymatic methods.

The search results indicate that researchers preparing 57Fe-labeled samples of ispH successfully employed techniques to maintain cluster integrity , suggesting that careful attention to these challenges can yield properly folded, active enzyme.

How can isotopic labeling be used to elucidate the reaction mechanism of recombinant Nitrosomonas europaea ispH?

Isotopic labeling provides powerful insights into the ispH reaction mechanism:

The search results specifically mention the use of 57Fe-labeled samples of ispH for spectroscopic studies, which enabled researchers to perform electron paramagnetic resonance (EPR), 57Fe electron-nuclear double resonance (ENDOR), and Mössbauer spectroscopy . This comprehensive spectroscopic approach allowed for a complete determination of the 57Fe hyperfine tensors for all four Fe ions in the [4Fe-4S] cluster, which is rarely reported in the literature and provides valuable insights into the electronic structure of the enzyme during catalysis.

What expression systems and conditions optimize the yield of active recombinant Nitrosomonas europaea ispH?

Based on approaches used for iron-sulfur proteins, optimal expression of recombinant Nitrosomonas europaea ispH likely requires:

• Host Selection:

  • E. coli BL21(DE3) or Rosetta(DE3) strains, which possess efficient iron-sulfur cluster assembly machinery

  • Consideration of codon optimization for Nitrosomonas europaea sequences

• Vector Design:

  • Inclusion of an affinity tag (His-tag) for purification

  • Addition of a cleavable linker if the tag affects enzyme activity

  • Strong but controllable promoter (T7 system)

• Culture Conditions:

  • Growth medium supplemented with iron sources (ferric ammonium citrate) and cysteine

  • Growth at 37°C until OD600 reaches 0.6-0.8, followed by temperature reduction to 18-20°C

  • Induction with low IPTG concentration (0.1-0.3 mM)

  • Extended expression time (16-20 hours) at lower temperature

• Anaerobic Considerations:

  • Growth in vessels with minimal headspace

  • Use of oxygen scavengers in media

  • Harvesting and processing under anaerobic conditions

While the search results don't provide specific details for Nitrosomonas europaea ispH expression, the successful preparation of 57Fe-labeled samples mentioned in result suggests that effective expression and purification protocols exist for this enzyme.

What is the recommended protocol for assaying ispH activity from Nitrosomonas europaea?

A comprehensive ispH activity assay protocol would include:

• Substrate Preparation:

  • Use of chemically synthesized or commercially available HMBPP

  • Verification of substrate purity by HPLC or mass spectrometry

• Electron Donation System Options:

  • Flavodoxin/flavodoxin reductase/NADPH system (most physiologically relevant)

  • Methyl viologen reduced with sodium dithionite (simpler alternative)

  • Dithionite alone (less physiological but effective for initial screening)

• Reaction Conditions:

  • Buffer: 100 mM HEPES or Tris, pH 7.5-8.0, containing 100 mM KCl and 5 mM MgCl2

  • Temperature: 30°C (typical for Nitrosomonas enzymes)

  • Strict anaerobic conditions using sealed vials or an anaerobic chamber

  • Inclusion of reducing agents (DTT or β-mercaptoethanol)

• Product Detection Methods:

  • HPLC analysis using a C18 reversed-phase column

  • LC-MS for sensitive detection and differentiation between IPP and DMAPP products

  • Analysis of product ratio (IPP:DMAPP) as an indicator of enzyme mechanism

• Data Analysis:

  • Determination of kinetic parameters (kcat, KM) under varying substrate concentrations

  • Assessment of effects of pH, temperature, and ionic strength on activity

The search results indicate that researchers have successfully studied ispH using a combination of spectroscopic techniques and activity assays , suggesting established protocols exist for measuring its activity.

How can site-directed mutagenesis be used to investigate the catalytic mechanism of Nitrosomonas europaea ispH?

Site-directed mutagenesis offers a powerful approach to dissect the catalytic mechanism of ispH:

• Target Residue Selection Based On:

  • Sequence conservation across ispH homologs

  • Structural information from crystallography or homology modeling

  • Proximity to the [4Fe-4S] cluster or substrate binding site

• Key Residues to Consider:

  • Conserved cysteines coordinating the [4Fe-4S] cluster

  • Residues potentially involved in substrate binding

  • Amino acids that may participate in proton transfer steps

• Mutation Design Strategy:

  • Conservative substitutions to probe specific functional aspects

  • Alanine scanning to identify essential residues

  • Introduction of non-native residues to alter electronic properties

• Analysis of Mutant Properties:

  • Activity assays to determine effects on catalysis

  • Spectroscopic studies (EPR, Mössbauer) to assess [4Fe-4S] cluster properties

  • Substrate binding studies

  • Product ratio (IPP:DMAPP) determination

• Mechanistic Interpretation:

  • Correlation of mutation effects with proposed catalytic steps

  • Development of a comprehensive catalytic model

The spectroscopic methods described in search result , particularly EPR and Mössbauer spectroscopy, would be invaluable for characterizing the effects of mutations on the [4Fe-4S] cluster properties and substrate interactions.

How do different electron donors affect the catalytic efficiency of recombinant Nitrosomonas europaea ispH?

The choice of electron donor significantly impacts ispH catalytic performance and experimental outcomes:

• Physiological vs. Artificial Electron Donors:

  • In vivo, electrons likely transfer via ferredoxin or flavodoxin systems

  • In vitro studies often use artificial donors like reduced methyl viologen or dithionite

  • The source of electrons can affect reaction rates and product distribution

• Redox Potential Considerations:

  • The [4Fe-4S] cluster in ispH cycles between +2 and +1 oxidation states during catalysis

  • Efficient reduction requires electron donors with appropriate redox potentials

  • Too negative potentials may cause over-reduction and enzyme inactivation

• Kinetic Implications:

  • The rate-limiting step can shift depending on the electron donation system

  • With physiological donors, electron transfer is often rate-limiting

  • With artificial donors, other steps like substrate binding or product release may become limiting

• Comparative Data:
  Research with other ispH enzymes suggests the following relationships:

  Electron Donor System	Relative Activity	Product Specificity	Notes
  Flavodoxin/FNR/NADPH	Lower	More selective	Most physiologically relevant
  Methyl viologen/dithionite	Higher	Less selective	Convenient for in vitro studies
  Dithionite alone	Variable	Less selective	Simple but less controlled

• Experimental Recommendations:

  • Characterize enzyme with multiple electron donation systems

  • Consider physiological relevance when interpreting kinetic data

  • Report standardized conditions for cross-laboratory comparison

What is known about the redox states of the [4Fe-4S] cluster during the catalytic cycle of ispH?

The [4Fe-4S] cluster in ispH cycles through multiple redox and ligation states during catalysis:

• Oxidized Resting State (Ox):

  • Contains a diamagnetic [4Fe-4S]2+ cluster

  • EPR-silent due to paired electrons

  • Mössbauer spectroscopy confirms the 2+ oxidation state

• Reduced State (Red):

  • Upon reduction, generates [4Fe-4S]+ with complex spin properties

  • Exhibits both S = 1/2 and S = 3/2 spin ground states

  • Detectable by EPR spectroscopy

• Substrate-Bound Intermediate (Int):

  • When reduced enzyme reacts with substrate, forms a transient paramagnetic species

  • Thought to contain a cluster-bound substrate-derived species

  • EPR properties suggest a 3+ iron-sulfur cluster oxidation state, confirmed by Mössbauer spectra

• Product-Bound State (Red+P):

  • Incubation of reduced enzyme with product (IPP) induces another paramagnetic species

  • Has S = 1/2 spin state with g-tensor typically associated with 3+ oxidation state

  • Mössbauer parameters show features typical for 2+ clusters, presenting an interesting mechanistic puzzle

These different states provide insight into the electron transfer events and substrate interactions occurring during catalysis, though full interpretation of the complex spectroscopic data requires careful analysis.

How do hypoxic conditions affect the expression and activity of ispH in Nitrosomonas europaea?

Nitrosomonas europaea undergoes significant physiological changes under hypoxic conditions, which likely impact ispH expression and activity:

• Transcriptional Regulation:

  • Nitrosomonas europaea shows a profound transcriptomic response when transitioned to hypoxic conditions

  • While direct evidence for ispH regulation is not provided in the search results, genes involved in key metabolic pathways show altered expression under oxygen limitation

• Metabolic Context:

  • Under hypoxia, Nitrosomonas europaea shifts to nitrifier denitrification, resulting in increased production of nitric oxide (NO) and nitrous oxide (N2O)

  • This metabolic shift alters the cellular redox balance, potentially affecting ispH function

  • Nitrogenous gases are formed through both enzymatic processes and chemical reactions involving key metabolites

• Potential Effects on IspH:

  • Altered availability of reducing equivalents for ispH activity

  • Changed expression levels to match metabolic demands

  • Possible interactions between reactive nitrogen species and the [4Fe-4S] cluster

  • Adaptation mechanisms to maintain essential isoprenoid biosynthesis under stress conditions

• Research Approaches:

  • Transcriptome analysis comparing ispH expression under aerobic vs. hypoxic conditions

  • Activity assays with cell extracts from cultures grown under different oxygen tensions

  • Purification and characterization of ispH from cells grown under hypoxic conditions

  • Investigation of potential post-translational modifications induced by hypoxia

Understanding how ispH functions under varying oxygen conditions would provide insights into Nitrosomonas europaea's metabolic adaptability in different environmental niches.

What is the relationship between ispH activity and the nitrogen metabolism in Nitrosomonas europaea?

While the search results don't directly address the relationship between ispH and nitrogen metabolism, we can consider potential connections based on Nitrosomonas europaea physiology:

• Metabolic Integration:

  • Nitrosomonas europaea derives energy from ammonia oxidation to nitrite via hydroxylamine as an intermediate

  • This process involves ammonia monooxygenase and hydroxylamine oxidoreductase (HAO)

  • The isoprenoid biosynthetic pathway (including ispH) likely competes with nitrogen metabolism for reducing equivalents

• Denitrification Connections:

  • Nitrosomonas europaea possesses denitrification enzymes, including nitric oxide reductase (Nor) encoded by the norCBQD gene cluster

  • The expression of denitrification enzymes occurs even under aerobic conditions

  • Both denitrification enzymes and iron-sulfur proteins like ispH are sensitive to oxygen levels

• Potential Regulatory Overlap:

  • Transcriptional regulators responding to nitrogen availability or redox state might coordinately regulate both pathways

  • Metabolic bottlenecks in either pathway could affect the other due to shared resources

  • Adaptation to environmental stresses likely requires coordinated regulation

• Functional Hypotheses:

  • Isoprenoids produced via the MEP pathway (involving ispH) may be important for membrane integrity during nitrogen stress

  • Specialized isoprenoid products might participate in signaling related to nitrogen metabolism

  • Optimal functioning of ammonia oxidation machinery might require specific isoprenoid-containing cofactors or membrane components

Further research specifically examining the cross-talk between isoprenoid biosynthesis and nitrogen metabolism would be valuable for understanding Nitrosomonas europaea's ecological adaptations.