Recombinant Xylella fastidiosa Ferrochelatase (hemH)

What is Xylella fastidiosa ferrochelatase (hemH) and what is its role in bacterial metabolism?

Xylella fastidiosa ferrochelatase (hemH) is the terminal enzyme in the heme biosynthesis pathway, catalyzing the insertion of ferrous iron (Fe²⁺) into protoporphyrin IX to form protoheme (heme B). This reaction represents the final step in heme biosynthesis and is critical for bacterial metabolism and survival. Bacterial ferrochelatases, including those from Xylella fastidiosa, function similarly to their eukaryotic counterparts but typically lack the [2Fe-2S] clusters found in human ferrochelatase .

In X. fastidiosa specifically, heme biosynthesis is essential for cytochrome formation and respiratory chain function, contributing to the bacterium's ability to survive in the nutrient-limited xylem environment of host plants. Unlike human ferrochelatase, which is membrane-associated, bacterial ferrochelatases are generally soluble enzymes, making them potentially more amenable to recombinant expression and purification.

How do I clone the hemH gene from Xylella fastidiosa for recombinant expression?

To clone the hemH gene from Xylella fastidiosa, you should follow these methodological steps:

• Primer design: Design specific primers based on the published hemH gene sequence from X. fastidiosa genomes. Include appropriate restriction sites for subsequent cloning into your expression vector. For improved specificity, consider using nested PCR approaches similar to those used for X. fastidiosa detection .

• DNA extraction: Extract genomic DNA from cultured X. fastidiosa using optimized protocols developed for this fastidious bacterium. The use of ionic exchange resin (Chelex 100) can increase DNA extraction efficiency as demonstrated for X. fastidiosa detection .

• PCR amplification: Amplify the hemH gene using high-fidelity polymerase. Initial denaturation at 94-95°C for 5 minutes, followed by 30-35 cycles of denaturation (94°C, 30s), annealing (55-60°C, 30s), and extension (72°C, 1 min per kb), with a final extension at 72°C for 7 minutes.

• Cloning strategy: Consider using Gateway cloning or traditional restriction enzyme-based cloning. For the latter, digest both the PCR product and expression vector with appropriate restriction enzymes, ligate, and transform into competent E. coli cells.

• Sequence verification: Always verify the cloned sequence to ensure no mutations were introduced during PCR amplification.

When selecting expression vectors, consider including affinity tags (His-tag, GST) to facilitate subsequent purification, and evaluate both N-terminal and C-terminal tag placements since tag position can affect enzyme activity.

What expression systems work best for producing recombinant Xylella fastidiosa ferrochelatase?

Several expression systems have been evaluated for recombinant bacterial ferrochelatases, with E. coli being the most commonly used for X. fastidiosa proteins. The optimal system depends on your specific research needs:

• E. coli BL21(DE3) and derivatives: These strains are recommended for their reduced protease activity and controlled induction via T7 RNA polymerase. For X. fastidiosa ferrochelatase, BL21(DE3)pLysS may help reduce basal expression if the protein shows toxicity.

• E. coli SHuffle: If disulfide bonds are critical for proper folding, this strain provides an oxidizing cytoplasmic environment.

• Cold-adapted expression: Induction at lower temperatures (15-25°C) often improves solubility of bacterial ferrochelatases.

• Codon optimization: The GC content of X. fastidiosa differs from E. coli, so codon optimization of the hemH gene may improve expression yields.

Expression vectors with tightly controlled promoters (T7, tac, or arabinose-inducible) allow for regulated expression, which is crucial for proteins that might be toxic to the host when overexpressed. Including iron supplementation (50-100 μM ferrous ammonium sulfate) in the culture medium can sometimes improve the yield of active ferrochelatase.

What are the typical yields of purified recombinant Xylella fastidiosa ferrochelatase?

Typical yields of purified recombinant X. fastidiosa ferrochelatase vary depending on the expression system and purification strategy employed. Based on similar bacterial ferrochelatases, researchers can expect:

• E. coli expression systems: 5-15 mg of purified protein per liter of culture under optimized conditions.

• Factors affecting yield:

  • Induction conditions (temperature, IPTG concentration, duration)

  • Cell lysis methods (sonication vs. chemical lysis)

  • Purification strategy (number of steps, buffer conditions)

  • Protein solubility and stability

• Optimization strategies:

  • Adding 5-10% glycerol to all buffers improves stability

  • Including 0.1-0.5 mM DTT or 2-mercaptoethanol helps prevent oxidation

  • Maintaining strict temperature control (4°C) during purification

  • Using protease inhibitors during initial extraction steps

For quantitative comparison across different preparation methods, specific activity (μmol product formed/min/mg protein) is a more reliable measure than total protein yield. When developing purification protocols, researchers should aim for >90% purity as assessed by SDS-PAGE while retaining enzymatic activity.

How can I optimize the purification protocol for recombinant Xylella fastidiosa ferrochelatase?

Optimizing purification of recombinant X. fastidiosa ferrochelatase requires careful consideration of protein properties and purification techniques:

• Affinity chromatography: For His-tagged constructs, immobilized metal affinity chromatography (IMAC) using Ni-NTA resin is most common. Critical parameters include:

  • Imidazole concentration in binding buffer (10-20 mM to reduce non-specific binding)

  • Imidazole concentration in elution buffer (gradient from 50-300 mM for optimal separation)

  • Flow rate (0.5-1 ml/min to ensure binding efficiency)

• Buffer optimization:

  • pH: Typically 7.5-8.0 works well for bacterial ferrochelatases

  • Salt concentration: 100-300 mM NaCl helps reduce non-specific interactions

  • Additives: 5-10% glycerol and 0.1-0.5 mM DTT improve stability

• Secondary purification:

  • Size exclusion chromatography to separate monomeric from aggregated protein

  • Ion exchange chromatography (typically Q Sepharose) as a polishing step

• Activity preservation:

  • Maintain reducing conditions throughout purification

  • Consider adding metal chelators (0.1 mM EDTA) in final dialysis buffer to remove any bound metals that might auto-oxidize and damage the enzyme

  • Avoid freeze-thaw cycles; store at 4°C for short term or flash-freeze aliquots in liquid nitrogen

• Quality control metrics:

  • Specific activity measurements at each purification step

  • SDS-PAGE and Western blotting to confirm identity and purity

  • Dynamic light scattering to assess monodispersity

The purification protocol should be validated by demonstrating that the final product retains enzymatic activity comparable to native ferrochelatase, with minimal contaminating proteins or enzymatic activities.

What assays can be used to measure the enzymatic activity of recombinant Xylella fastidiosa ferrochelatase?

Several assays can be employed to measure ferrochelatase activity, each with specific advantages:

• Spectrophotometric continuous assay:

  • Principle: Monitors decrease in protoporphyrin IX absorbance or increase in metalloporphyrin formation

  • Wavelengths: Typically 408 nm for substrate disappearance or 421 nm for product formation

  • Advantages: Real-time kinetics, relatively simple setup

  • Limitations: Lower sensitivity, potential interference from protein absorbance

• Fluorometric assay:

  • Principle: Measures the decrease in protoporphyrin IX fluorescence (excitation 405 nm, emission 630 nm)

  • Advantages: 10-100× more sensitive than spectrophotometric methods

  • Limitations: Quenching effects, requires careful calibration

• HPLC-based assay:

  • Principle: Separates substrate and product based on hydrophobicity

  • Detection: UV-visible or fluorescence detection

  • Advantages: High specificity, can resolve multiple reaction products

  • Limitations: Equipment-intensive, discontinuous measurements

• Coupled enzyme assays:

  • Principle: Links ferrochelatase activity to more easily detectable reactions

  • Examples: Coupling with heme oxygenase to monitor heme degradation

  • Advantages: Can amplify weak signals

  • Limitations: Multiple variables affect results, complex optimization

Standard reaction conditions typically include:

• 50-100 mM Tris-HCl or HEPES buffer, pH 7.5-8.0

• 0.5-5 μM protoporphyrin IX (dissolved in minimal DMSO, <1% final concentration)

• 1-50 μM ferrous iron (as ferrous ammonium sulfate with reducing agent)

• 0.1-1 mg/ml bovine serum albumin to stabilize the enzyme

• 1-5 mM β-mercaptoethanol or 0.5-2 mM DTT as reducing agent

When reporting activity data, always include specific activity, substrate concentrations, temperature, and buffer composition to allow for comparison across studies.

How does the substrate specificity of Xylella fastidiosa ferrochelatase compare to other bacterial ferrochelatases?

Bacterial ferrochelatases typically show broader substrate specificity than their eukaryotic counterparts. While specific data for X. fastidiosa ferrochelatase substrate specificity is limited, comparison with other bacterial ferrochelatases reveals important patterns:

X. fastidiosa ferrochelatase likely shares the general characteristics of other Gram-negative bacterial ferrochelatases, but definitive comparisons require direct experimental determination of substrate preferences and kinetic parameters.

What structural features distinguish Xylella fastidiosa ferrochelatase from other bacterial and eukaryotic ferrochelatases?

The structural features of X. fastidiosa ferrochelatase can be inferred from comparison with known bacterial and eukaryotic ferrochelatase structures, highlighting several distinctive characteristics:

These structural differences have significant implications for enzyme function and regulation, potentially contributing to the unique physiological adaptations of X. fastidiosa to its host environment. Understanding these distinctions can guide the development of structure-based inhibitors targeting bacterial ferrochelatases while sparing human enzymes.

How does ferrochelatase activity contribute to Xylella fastidiosa pathogenicity in different host plants?

Ferrochelatase activity likely plays a multifaceted role in X. fastidiosa pathogenicity across different host plants:

• Iron acquisition and metabolism:

  • Heme biosynthesis is essential for cytochrome formation and respiratory function

  • In the xylem environment where X. fastidiosa colonizes, iron availability is limited, making efficient ferrochelatase activity crucial for bacterial survival

  • X. fastidiosa subspecies show host specificity patterns , which may partially relate to their ability to adapt to iron acquisition challenges in different plant species

• Oxidative stress response:

  • Heme-containing proteins are critical for detoxifying reactive oxygen species produced during plant defense responses

  • Ferrochelatase activity may be upregulated during oxidative stress conditions, similar to observations in other bacterial pathogens

  • Host plants with stronger oxidative bursts may exert selective pressure on ferrochelatase function

• Biofilm formation and virulence:

  • Heme-dependent sensors and regulators influence biofilm formation in many bacteria

  • X. fastidiosa pathogenicity depends heavily on biofilm formation within xylem vessels

  • Preliminary transcriptomic analyses reveal complex gene expression patterns during infection , potentially including hemH regulation

• Host-specific adaptations:

  • X. fastidiosa shows varying degrees of virulence in different hosts, from severe disease to commensal relationships

  • Ferrochelatase activity may be differentially regulated based on host-specific signals

  • The compatibility between xylem composition and X. fastidiosa-secreted enzymes mediates disease onset and progression

• Potential research approaches:

  • Generate hemH knockout or conditional mutants to assess virulence in different host plants

  • Compare hemH expression levels during infection of susceptible versus resistant hosts

  • Evaluate ferrochelatase activity under conditions mimicking different host xylem environments

While direct evidence linking ferrochelatase activity to X. fastidiosa host range and pathogenicity is still emerging, the central role of heme in bacterial metabolism and stress response suggests this enzyme contributes significantly to the pathogen's ability to colonize and cause disease in diverse plant hosts.

What is the role of ferrochelatase in Xylella fastidiosa's adaptation to different environmental conditions?

Ferrochelatase likely plays a key role in X. fastidiosa's adaptation to varying environmental conditions:

• Temperature adaptation:

  • X. fastidiosa encounters temperature fluctuations in different hosts and geographic regions

  • Ferrochelatase activity may be modulated to maintain optimal heme biosynthesis across temperature ranges

  • Transcriptomic studies suggest temperature-dependent regulation of metabolic genes in X. fastidiosa

• Nutrient limitation responses:

  • Xylem sap is a nutrient-poor environment with variable iron availability

  • Ferrochelatase expression and activity may be upregulated under iron-limited conditions

  • Alternative metal ions might be incorporated under extreme iron limitation, producing modified heme molecules

• Oxidative and nitrosative stress adaptation:

  • Plants produce reactive oxygen and nitrogen species during defense responses

  • Heme-containing detoxification enzymes (catalases, peroxidases) protect against these stresses

  • Ferrochelatase activity may increase to support enhanced production of protective heme proteins

• Biofilm versus planktonic lifestyle transitions:

  • X. fastidiosa transitions between biofilm and planktonic growth phases during infection

  • These transitions involve substantial metabolic reprogramming

  • Preliminary transcriptomic data indicates differential expression of metabolic genes during these transitions

• Adaptations to different plant hosts:

  • X. fastidiosa subspecies show associations with specific host ranges

  • Ferrochelatase activity may be optimized for the specific microenvironments provided by different host xylem compositions

  • The fine balance between commensalism and parasitism in different hosts might involve modulation of basic metabolic functions including heme biosynthesis

Experimental approaches to investigate these adaptations could include:

• Measuring ferrochelatase activity under various stress conditions (temperature, pH, oxidative stress)

• Analyzing hemH expression in different growth phases and host environments

• Comparing ferrochelatase kinetic parameters among X. fastidiosa strains with different host preferences

Can inhibitors of Xylella fastidiosa ferrochelatase be developed as potential antimicrobial agents?

The development of X. fastidiosa ferrochelatase inhibitors as antimicrobial agents presents several opportunities and challenges:

• Target validation considerations:

  • Ferrochelatase is essential for heme biosynthesis in most bacteria

  • Genetic studies in related bacteria confirm hemH as a potential antimicrobial target

  • X. fastidiosa's dependence on respiratory metabolism in the xylem environment makes heme biosynthesis particularly critical

• Inhibitor design strategies:

  • Substrate analogs: Modified porphyrins that compete with protoporphyrin IX

  • Metal-binding inhibitors: Compounds that interfere with iron coordination

  • Allosteric inhibitors: Molecules binding outside the active site to alter enzyme conformation

  • Transition-state analogs: Compounds mimicking the reaction transition state

• Selectivity considerations:

  • Structural differences between bacterial and plant/human ferrochelatases can be exploited

  • Absence of [2Fe-2S] clusters in bacterial ferrochelatases versus human enzyme

  • Different active site architectures affect inhibitor binding profiles

• Delivery challenges in planta:

  • Inhibitors must reach X. fastidiosa in xylem vessels

  • Compound stability in xylem sap environment

  • Plant uptake and systemic distribution requirements

  • Potential phytotoxicity concerns

• Screening methodology:

  Screening Approach	Advantages	Limitations	Example Assay Conditions
  In vitro enzyme assays	Direct measure of inhibition, quantitative	May not predict in vivo efficacy	HEPES buffer pH 7.5, 1-5 μM enzyme, 0.5-10 μM substrate
  Whole-cell growth inhibition	Assesses membrane permeability, metabolism	Less specific, multiple targets	PWG media, 10^6-10^7 CFU/mL, 24-96h incubation
  Ex vivo xylem model	More physiologically relevant	Lower throughput, complex setup	Artificial xylem fluid, microfluidic chambers

• Resistance development risk:

  • Single target inhibition may lead to resistance

  • Combination approaches targeting multiple steps in heme biosynthesis may reduce resistance risk

  • Structure-activity relationship studies to optimize inhibitor properties

While significant research would be required, the essential nature of ferrochelatase and the structural differences between bacterial and eukaryotic enzymes make this a promising target for developing novel antimicrobials against X. fastidiosa.

How does the expression of ferrochelatase change during Xylella fastidiosa biofilm formation?

Biofilm formation is a critical aspect of X. fastidiosa pathogenicity, and ferrochelatase expression and activity likely undergo significant changes during this process:

• Temporal expression patterns:

  • Initial attachment phase: Potential upregulation to support increased energy demands

  • Mature biofilm: Possible differential expression in various biofilm regions

  • Dispersion phase: Changes to support transition to planktonic lifestyle

  • Recent pilot RNA-seq studies of X. fastidiosa in planta provide initial insights into gene expression dynamics during infection

• Spatial expression heterogeneity:

  • Cells at biofilm periphery may show different ferrochelatase expression than interior cells

  • Oxygen gradients within biofilms could influence heme biosynthesis regulation

  • Nutrient availability differences throughout biofilm structure

• Regulatory mechanisms:

  • Quorum sensing systems likely influence hemH expression

  • Two-component systems responding to environmental cues

  • Small regulatory RNAs may fine-tune expression levels

  • Iron-responsive regulators could coordinate ferrochelatase with iron acquisition systems

• Metabolic adaptations:

  • Shift from planktonic to biofilm growth involves substantial metabolic reprogramming

  • Heme requirements may change with altered respiratory activity in biofilm state

  • Energy conservation strategies in mature biofilms might affect heme biosynthesis rates

• Experimental approaches to study expression changes:

  • Transcriptomic analysis (RNA-Seq) comparing planktonic versus biofilm cells

  • Fluorescent reporter fusions to monitor real-time expression in biofilm development

  • Quantitative proteomics to assess protein-level changes

  • Enzyme activity assays from cells harvested at different biofilm development stages

Understanding these expression patterns could provide insights into potential intervention points for disrupting biofilm formation by targeting ferrochelatase activity at critical developmental stages. The recent development of transcriptomic analysis methods for X. fastidiosa presents new opportunities to explore these expression dynamics in detail.

What are the most effective methods for site-directed mutagenesis of Xylella fastidiosa ferrochelatase?

Site-directed mutagenesis of X. fastidiosa ferrochelatase can be accomplished through several approaches, each with specific advantages:

When planning mutagenesis studies of X. fastidiosa ferrochelatase, consider creating sets of mutations targeting: (1) metal coordination residues, (2) substrate binding pocket residues, (3) potential regulatory sites, and (4) residues at interfaces with potential protein partners.

How can I develop a high-throughput screening assay for inhibitors of Xylella fastidiosa ferrochelatase?

Developing a high-throughput screening (HTS) assay for X. fastidiosa ferrochelatase inhibitors requires careful assay design and optimization:

• Assay format selection:

  • Fluorescence-based assays: Measure decrease in protoporphyrin IX fluorescence

    • Excitation: 405 nm, Emission: 630 nm

    • Advantages: High sensitivity, compatibility with 384/1536-well formats

    • Limitations: Potential fluorescence interference from compounds

  • Coupled enzyme assays: Link ferrochelatase activity to a detectable output

    • Example: Couple with heme oxygenase and biliverdin reductase

    • Advantages: Signal amplification, potential for absorbance readout

    • Limitations: More complex, multiple enzymes increase variables

• Assay optimization parameters:

  • Enzyme concentration: Typically 10-50 nM (optimize for signal window)

  • Substrate concentrations: Set near K_m (typically 1-5 μM protoporphyrin IX, 5-20 μM Fe²⁺)

  • Buffer composition: HEPES or Tris buffer (pH 7.5-8.0), 100-150 mM NaCl, 0.1-0.5 mM DTT

  • Reaction time: 15-30 minutes (must be in linear range)

  • Plate format: 384-well black plates for fluorescence assays

  • Controls: DMSO-only (100% activity), known inhibitors or no-enzyme (0% activity)

• Assay validation metrics:

  • Z' factor: Target >0.5 for robust screening

  • Signal-to-background ratio: >4 recommended

  • Coefficient of variation: <10% for reliable results

  • DMSO tolerance: Test 0.1-1% DMSO to ensure minimal impact on enzyme activity

  • Day-to-day reproducibility: <15% variation in control wells

• Screening workflow design:

  • Primary screen: Single concentration (10-20 μM) of compounds, identify hits with >50% inhibition

  • Confirmation screen: Retest primary hits in duplicate/triplicate

  • Dose-response determination: 8-10 point curves for confirmed hits, determine IC₅₀ values

  • Counter-screen: Test against related enzymes to assess selectivity

  • Orthogonal assays: Confirm activity using alternative assay format

• Addressing screening challenges:

  • Compound interference: Include control wells with compounds but no enzyme reaction

  • Enzyme stability: Prepare fresh enzyme regularly, consider stabilizing additives

  • Redox-sensitive compounds: Include reducing agents in assay buffer

  • Aggregation-based inhibitors: Include 0.01-0.05% detergent (Triton X-100) to reduce false positives

By carefully optimizing these parameters, researchers can develop a robust HTS assay for identifying potential inhibitors of X. fastidiosa ferrochelatase with good sensitivity and specificity.

What are the best approaches for studying the interaction between Xylella fastidiosa ferrochelatase and other proteins in the heme biosynthesis pathway?

Several complementary approaches can be employed to study protein-protein interactions involving X. fastidiosa ferrochelatase:

• Co-immunoprecipitation (Co-IP) studies:

  • Methodology: Express tagged ferrochelatase in X. fastidiosa or heterologous system, isolate using specific antibodies or tag-based pulldown, identify interacting partners via mass spectrometry

  • Advantages: Can detect native complexes under physiological conditions

  • Limitations: May miss transient interactions, requires efficient antibodies or functional tagged proteins

  • Optimization: Use reversible crosslinking (0.5-1% formaldehyde) to capture transient interactions

• Bacterial two-hybrid systems:

  • Methodology: Create fusion proteins with split reporter domains, interaction reconstitutes reporter activity

  • Advantages: In vivo detection, suitable for large-scale screening

  • Limitations: May yield false positives/negatives, fusion proteins may alter interaction dynamics

  • Systems: BACTH (bacterial adenylate cyclase-based), LexA-based, or λ repressor-based approaches

• Surface plasmon resonance (SPR):

  • Methodology: Immobilize purified ferrochelatase on sensor chip, flow potential interacting proteins, measure binding kinetics

  • Advantages: Real-time, label-free detection, provides quantitative binding parameters (kon, koff, KD)

  • Limitations: Requires highly purified proteins, immobilization may affect binding properties

  • Parameters: Typical flow rates 20-50 μL/min, analyte concentrations spanning 0.1-10× KD

• Microscale thermophoresis (MST):

  • Methodology: Label one protein, mix with varying concentrations of unlabeled partner, measure changes in thermophoretic mobility

  • Advantages: Low sample consumption, works in complex biological matrices

  • Limitations: Requires protein labeling, potential interference from buffer components

  • Sensitivity: Can detect interactions with KD from nM to mM range

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS):

  • Methodology: Compare deuterium uptake patterns of ferrochelatase alone versus in complex with partners

  • Advantages: Maps interaction interfaces, detects conformational changes upon binding

  • Limitations: Complex data analysis, requires specialized equipment

  • Resolution: Can identify specific regions/residues involved in interactions

When specifically investigating interactions within the heme biosynthesis pathway, consider:

• Testing interactions with glutamyl-tRNA reductase, the pathway's rate-limiting enzyme

• Examining potential interactions with iron transport proteins that may channel substrate

• Investigating associations with regulatory proteins that might coordinate pathway activity

A combination of these approaches provides complementary data to build a comprehensive picture of the protein interaction network involving X. fastidiosa ferrochelatase.

How can I measure the iron-binding kinetics of recombinant Xylella fastidiosa ferrochelatase?

Accurately measuring iron-binding kinetics of X. fastidiosa ferrochelatase requires specialized techniques to address the challenges of working with redox-sensitive iron:

• Isothermal titration calorimetry (ITC):

  • Methodology: Titrate ferrous iron solution into protein sample, measure heat changes

  • Experimental setup:

    • Protein concentration: 10-50 μM in degassed buffer

    • Iron solution: 0.5-2 mM ferrous ammonium sulfate with reducing agent

    • Buffer: 50 mM HEPES, pH 7.5, 150 mM NaCl, 1-5 mM TCEP or 2-5 mM sodium dithionite

    • Temperature: 25°C

  • Data analysis: Fit to appropriate binding model (typically one-site or sequential binding)

  • Advantages: Direct measurement of thermodynamic parameters (ΔH, ΔS, KD)

  • Limitations: High protein consumption, potential oxidation of Fe²⁺

• Fluorescence spectroscopy:

  • Methodology: Monitor changes in intrinsic tryptophan fluorescence upon iron binding

  • Experimental setup:

    • Excitation wavelength: 295 nm (specific for tryptophan)

    • Emission scan: 310-400 nm

    • Protein concentration: 1-5 μM

    • Titrate with 0.1-50 μM ferrous iron

  • Data analysis: Plot fluorescence intensity change versus iron concentration, fit to binding equation

  • Advantages: Sensitive, low protein requirement

  • Limitations: Indirect measurement, may be affected by protein conformation changes

• Stopped-flow spectroscopy:

  • Methodology: Rapidly mix enzyme and iron, monitor spectral changes over millisecond-second timescale

  • Experimental setup:

    • Absorbance detection at 330-350 nm (Fe²⁺ binding region)

    • Temperature control at 25°C

    • Anaerobic conditions essential

  • Data analysis: Fit to appropriate kinetic models (single exponential, two-step binding)

  • Advantages: Measures association/dissociation rates directly

  • Limitations: Complex data analysis, specialized equipment required

• Electron paramagnetic resonance (EPR):

  • Methodology: Monitor changes in EPR spectra upon binding of paramagnetic Fe²⁺/Fe³⁺

  • Experimental considerations:

    • Low temperature measurements (liquid nitrogen or helium)

    • Precise control of oxidation state

  • Advantages: Direct observation of iron environment

  • Limitations: Requires specialized equipment, complex data interpretation

• Practical considerations for all methods:

  • Maintain strict anaerobic conditions to prevent Fe²⁺ oxidation (glove box or Schlenk techniques)

  • Include control experiments with metal chelators (EDTA) to verify specific binding

  • Compare binding of other divalent metals (Zn²⁺, Co²⁺) for selectivity analysis

  • Account for potential non-specific binding to surface residues

By applying these techniques with appropriate controls, researchers can obtain comprehensive kinetic and thermodynamic parameters for iron binding to X. fastidiosa ferrochelatase, providing insights into its catalytic mechanism.