Recombinant Ferredoxin-2

Basic Research Questions

• What is Ferredoxin-2 and what is its role in mitochondrial function?

  Ferredoxin-2 (FDX2) is a mitochondrial [2Fe-2S] cluster-containing protein that serves as an essential electron donor in the de novo assembly of iron-sulfur (FeS) clusters. FDX2 functions as a component of the mitochondrial iron-sulfur cluster (ISC) assembly machinery, working alongside the scaffold protein ISCU2, the cysteine desulfurase subcomplex NFS1-ISD11-ACP1, and the allosteric activator frataxin (FXN) .

  Unlike its paralog Ferredoxin-1 (FDX1/adrenodoxin), which primarily functions in steroidogenesis, FDX2 is crucially involved in both [2Fe-2S] and [4Fe-4S] cluster biosynthesis . This essential role makes FDX2 fundamental to numerous cellular processes dependent on FeS proteins, including respiration, DNA synthesis, and protein translation. Mutations in FDX2 are associated with iron accumulation in mitochondria and linked to several rare human diseases, including neurodegenerative conditions .

• How can researchers express and purify functional recombinant Ferredoxin-2?

  Successful recombinant expression and purification of functional FDX2 requires strategic approaches to ensure proper [2Fe-2S] cluster incorporation. Based on research protocols, the following methods have proven effective:

  Expression Systems:

  • Escherichia coli is the predominant heterologous expression system for FDX2 production

  • Expression vectors providing strong promoter control (such as T7) optimize protein yield

  • The mature protein sequence (without mitochondrial targeting sequence) should be used

  Expression Strategy:

  • FDX2 can be expressed as a fusion protein to enhance solubility and facilitate purification

  • Expression at lower temperatures (16-25°C) often improves proper folding and [2Fe-2S] cluster incorporation

  • Supplementation of the growth medium with iron salts and incorporation of an anaerobic environment during late expression phases can enhance cluster assembly

  Purification Protocol:

  • Initial clarification through centrifugation following cell lysis

  • Sequential chromatography typically involving:
    a) Affinity chromatography (Ni-NTA for His-tagged proteins)
    b) Ion exchange chromatography (typically anion exchange)
    c) Size exclusion chromatography for final polishing

  • All purification steps should ideally be performed under anaerobic or low-oxygen conditions

  Successful expression typically yields a pink/reddish-colored protein solution, characteristic of [2Fe-2S] cluster-containing ferredoxins .

• How can the proper incorporation and functionality of the [2Fe-2S] cluster in recombinant FDX2 be verified?

  Multiple complementary analytical techniques should be employed to verify proper [2Fe-2S] cluster incorporation and functionality:

  Spectroscopic Analysis:

  • UV-visible absorption spectroscopy: Properly folded FDX2 with intact [2Fe-2S] clusters shows characteristic absorption peaks with patterns distinctive of [2Fe-2S] ferredoxins

  • Circular dichroism (CD) spectroscopy: Provides information about both protein secondary structure and [2Fe-2S] cluster environment

  • Electron paramagnetic resonance (EPR) spectroscopy: The reduced [2Fe-2S]¹⁺ cluster is EPR-active, allowing confirmation of proper cluster coordination environment

  Functional Verification:

  • Electron transfer capacity: Assess reducibility by ferredoxin reductase (FDXR) using NADPH as electron donor

  • Functional assays: CD spectroscopy-based [2Fe-2S] cluster synthesis assays can monitor FDX2's ability to support cluster formation on ISCU2, measuring absorption at 431 nm

  Biochemical Characterization:

  • Redox potential determination: Provides information about the thermodynamic properties of the [2Fe-2S] cluster

  • Iron and sulfur content determination: Ideally showing a 2:2 Fe:S stoichiometry per protein monomer

• What are the key structural and functional differences between Ferredoxin-2 and Ferredoxin-1?

  Despite their sequence similarity as [2Fe-2S] cluster-containing proteins, FDX1 and FDX2 exhibit critical differences:

  Feature	Ferredoxin-1 (FDX1)	Ferredoxin-2 (FDX2)
  Primary function	Steroidogenesis, cytochrome P450 reduction	Iron-sulfur cluster biogenesis
  Tissue expression	Highly expressed in adrenal gland, kidney, testes	Ubiquitously expressed across tissues
  Specific structural features	Lacks C-terminal motif found in FDX2	Contains distinctive conserved C-terminus
  Electron acceptors	Efficiently reduces mitochondrial cytochromes P450	Transfers electrons to ISC machinery
  Biochemical specificity	Unable to efficiently support Fe/S protein biogenesis	Unable to efficiently reduce cytochromes P450

  Quantitative analysis of tissue distribution found that Fdx1 and Fdx2 were present in HeLa cells at approximately 90 and 40 ng/mg cell protein, respectively . While both ferredoxins can accept electrons from NADPH via ferredoxin reductase (FDXR), they exhibit remarkable substrate specificity in their downstream electron transfer pathways.

• What experimental approaches are used to study FDX2's role in iron homeostasis?

  FDX2 deficiency directly impacts cellular iron homeostasis through impaired Fe/S protein biogenesis. Researchers employ several approaches to study this relationship:

  Cellular Models:

  • RNA interference or CRISPR-based gene editing to deplete FDX2

  • Complementation studies with wild-type or mutant FDX2 variants

  • Cell lines with inducible FDX2 expression for temporal studies

  Iron Homeostasis Assessment:

  • Monitoring iron regulatory proteins (IRPs): FDX2 deficiency alters IRP1 and IRP2 mRNA binding activity

  • Measuring cellular iron uptake: FDX2 depletion increases iron uptake mechanisms

  • Analyzing mitochondrial iron accumulation: FDX2 deficiency leads to mitochondrial iron overload

  Functional Readouts:

  • Activity assays for Fe/S-dependent enzymes across cellular compartments

  • Quantification of iron-responsive element (IRE)-containing transcripts

  • Assessment of oxidative stress parameters associated with iron dysregulation

  These approaches have established that FDX2 deficiency has a severe impact on cellular iron homeostasis through impaired Fe/S protein biogenesis, leading to increased cellular iron uptake and pathological iron accumulation in mitochondria .

Advanced Research Questions

• What are the structural determinants that govern FDX2 interaction with the core ISC complex?

  Cryo-EM structures, combined with biochemical studies, have revealed several key structural determinants mediating FDX2 interaction with the core ISC complex:

  Two-Stage Binding Model:
  FDX2 can bind the core ISC complex in two distinct conformations :

  a) Distal Conformation:

  • FDX2's helix F interacts electrostatically with an arginine patch on NFS1

  • The [2Fe-2S] cluster of FDX2 remains relatively distant from the ISCU2 assembly site

  • Represents an initial recognition and binding state

  b) Proximal Conformation:

  • Electrostatic interaction between FDX2 and NFS1 tightens

  • The FDX2-specific C-terminus binds to NFS1

  • The [2Fe-2S] cluster moves closer to the ISCU2 FeS cluster assembly site

  • Facilitates rapid electron transfer for FeS cluster assembly

  Critical Interaction Elements:

  • The C-terminal region of FDX2 binds specifically to NFS1 in the proximal conformation

  • Residue Asn175 of FDX2 forms hydrogen bonds with Ser385 of NFS1

  • Salt bridges form between FDX2 and NFS1 in both binding conformations

  Competitive Binding Dynamics:

  • FDX2 and frataxin (FXN) compete for overlapping binding sites on the core ISC complex

  • This competition influences the dynamics and efficiency of FeS cluster assembly

  These structural determinants collectively enable the specific binding of FDX2 to the core ISC complex and facilitate electron transfer necessary for FeS cluster assembly.

• What is the significance of the conserved C-terminus of Ferredoxin-2 in its function?

  The conserved C-terminus of FDX2 plays several crucial roles in its function and specificity:

  Structural Basis of Specificity:

  • The C-terminal sequence distinguishes "type II ferredoxin" (FDX2) from type-I adrenodoxin (FDX1) and fungal-type eukaryotic [2Fe-2S] ferredoxins

  • This structural difference contributes to FDX2's specialization for FeS cluster biosynthesis

  Interaction with the ISC Complex:

  • In the proximal conformation, the C-terminus binds to NFS1

  • Key residues include Asn175, which forms hydrogen bonds with Ser385 of NFS1

  • This interaction appears important for the transition from distal to proximal binding

  Effect on Cluster Assembly Rates:
  Truncation experiments with variants lacking C-terminal residues revealed unexpected results:

  • All truncated variants (ΔC1, ΔC5, ΔC10, ΔC12) could generate wild-type amounts of [2Fe-2S] clusters

  • ΔC1, ΔC5, and ΔC10 variants showed up to fourfold higher cluster assembly rates than wild-type FDX2 under standard conditions (5 μM)

  • The ΔC12 variant showed a different kinetic profile with only 1.5-fold higher rates

  Site-Specific Mutational Evidence:

  • The FDX2-N175A mutant (replacing Asn with Ala) showed reduced efficiency in [2Fe-2S] cluster assembly at standard concentrations

  • FDX2-N175A required higher concentrations to achieve efficient cluster assembly, suggesting this residue is important but not essential

  Though not absolutely essential for in vitro [2Fe-2S] cluster assembly, the conserved C-terminus fine-tunes FDX2's interaction with the core ISC complex and influences the efficiency of FeS cluster biosynthesis.

• How does FDX2 compete with frataxin (FXN) for binding to the core ISC complex?

  FDX2 and frataxin (FXN) exhibit a competitive relationship for binding to the core ISC complex that dynamically regulates FeS cluster assembly:

  Structural Basis of Competition:

  • Cryo-EM structures show that FDX2 and FXN binding sites on the core ISC complex partially overlap

  • This creates a physical basis for competition where simultaneous binding of both proteins at their optimal positions may be restricted

  Concentration-Dependent Effects:

  • FDX2 shows a bell-shaped concentration-dependence curve for [2Fe-2S] cluster synthesis:

    • Maximum rate occurs at approximately 1-2 μM FDX2

    • Rates decrease at higher FDX2 concentrations

  • FXN also exhibits bell-shaped concentration-dependence:

    • Maximum rate above 1 μM FXN

    • Little change up to 10 μM FXN

    • Substantial drop at 20 μM FXN

  Relative Binding Dynamics:

  • FXN requires much higher concentrations (>10 μM) to decrease cluster synthesis rates

  • This suggests FXN is a comparatively weaker competitor under turnover conditions

  • Optimal relative concentrations of both proteins are required for maximum synthesis rates

  Regulatory Implications:

  • The competition likely serves as a regulatory mechanism for coordinating the multiple steps of FeS cluster assembly

  • Alterations in the balance between FDX2 and FXN could affect the efficiency of FeS cluster biosynthesis

  • This competitive relationship has particular relevance for understanding Friedreich's ataxia, where decreased FXN function leads to neurodegeneration

  This competition suggests that FeS cluster assembly involves a precisely coordinated sequence of protein interactions rather than simultaneous binding of all components.

• What spectroscopic methods are most effective for analyzing electron transfer in the context of FDX2-mediated FeS cluster assembly?

  Multiple spectroscopic techniques provide complementary insights into FDX2-mediated electron transfer:

  UV-Visible Absorption Spectroscopy:

  • Monitors redox state changes of FDX2's [2Fe-2S] cluster

  • Tracks cluster formation on ISCU2 during assembly

  • Can be performed in real-time to follow reaction progression

  • Particularly useful at wavelengths characteristic of [2Fe-2S] clusters (typically 330-460 nm)

  Circular Dichroism (CD) Spectroscopy:

  • Provides distinctive signals for [2Fe-2S] clusters in different environments

  • Used to monitor [2Fe-2S] cluster formation on ISCU2 at 431 nm

  • Can detect conformational changes accompanying electron transfer

  • Successfully employed in tracking FDX2-dependent reduction and cluster assembly

  Electron Paramagnetic Resonance (EPR) Spectroscopy:

  • Detects paramagnetic species including reduced [2Fe-2S]¹⁺ clusters

  • Can identify distinct intermediates during electron transfer

  • Provides detailed electronic structure information

  • Successfully used to characterize [2Fe-2S] cluster in recombinant ferredoxins

  Time-Resolved Techniques:

  • Stopped-flow spectroscopy: Measures rapid electron transfer kinetics

  • Rapid freeze-quench EPR: Captures transient intermediates

  • These approaches are particularly valuable for studying the sequential steps in electron transfer

  For effective experimental design, researchers should combine multiple spectroscopic methods with kinetic analyses, varying protein concentrations to identify rate-limiting steps and competition effects as demonstrated in studies with FDX2, FXN, and the core ISC complex .

• How do mutations in the C-terminal region of FDX2 affect its function in FeS cluster assembly?

  The C-terminal region of FDX2 has been extensively studied through mutational analysis, revealing nuanced effects on FeS cluster assembly:

  Truncation Effects on Cluster Assembly:

  FDX2 Variant	Cluster Formation Capability	Relative Rate (vs. WT)	Concentration Dependence
  Wild-type	Complete	1× (reference)	Bell-shaped curve with max at 1-2 μM
  ΔC1	Complete	Up to 4× higher	Similar to ΔC5/ΔC10
  ΔC5	Complete	Up to 4× higher	Similar to ΔC1/ΔC10
  ΔC10	Complete	Up to 4× higher	Similar to ΔC1/ΔC5
  ΔC12	Complete	~1.5× higher	Distinct pattern requiring higher concentration

  Site-Specific Mutations:

  • FDX2-N175A (mutation of Asn175 to Ala):

    • Maintained ability to support [2Fe-2S] cluster assembly

    • Required higher concentrations for efficient function

    • Showed kinetic behavior similar to the ΔC12 variant

    • Demonstrates the importance of Asn175 in FDX2 function

  Mechanistic Insights:

  • C-terminal residues influence the transition between distal and proximal binding conformations

  • The unexpected increase in activity with certain truncations suggests complex regulatory roles

  • The salt bridge formed between Asp179 (FDX2) and Arg393 (NFS1) appears to have minor influence on synthesis rates

  • Asn175 forms hydrogen bonds with Ser385 of NFS1 in the proximal conformation, playing a key role in positioning FDX2 for efficient electron transfer

  Evolutionary Considerations:

  • The C-terminal sequence distinguishes "type II ferredoxin" (FDX2) from type-I adrenodoxin (FDX1)

  • Fungal-type ferredoxins (S. cerevisiae Yah1, C. thermophilum Yah1) with different C-termini can also support in vitro [2Fe-2S] cluster synthesis

  These findings demonstrate that while the C-terminus is not absolutely essential for basic FDX2 function, it serves to optimize interaction dynamics with the core ISC complex and fine-tune the efficiency of FeS cluster assembly.

• What experimental approaches best characterize the two-stage binding model of FDX2 to the core ISC complex?

  The two-stage binding model (distal and proximal conformations) of FDX2 to the core ISC complex requires multifaceted experimental approaches:

  Structural Determination:

  • Cryo-electron microscopy (cryo-EM) has been instrumental in directly visualizing both conformational states

  • Hydrogen-deuterium exchange mass spectrometry (HDX-MS) can map regions with different solvent accessibility in each conformation

  • Cross-linking mass spectrometry can identify distance constraints between interacting residues

  Mutational Analysis with Functional Readouts:

  • Site-directed mutagenesis targeting key residues in:

    • Electrostatic interfaces (helix F region interacting with NFS1)

    • C-terminal region critical for transition to proximal conformation

    • The Asn175 position shown to be important for interaction with NFS1

  • Functional assessment using:

    • CD spectroscopy-based [2Fe-2S] cluster synthesis assays

    • Concentration-dependent activity measurements

    • Competition assays with FXN

  Kinetic and Thermodynamic Studies:

  • Binding affinity measurements using surface plasmon resonance or isothermal titration calorimetry

  • Pre-steady-state kinetics to resolve transitions between binding states

  • Electron transfer rate measurements in different conformational states

  Computational Approaches:

  • Molecular dynamics simulations of the transition between distal and proximal states

  • Calculation of electrostatic interaction energies

  • Modeling of electron transfer pathways in both conformations

  Distance Measurements:

  • FRET-based approaches to measure distances between labeled components

  • EPR-based distance measurements (DEER/PELDOR) for paramagnetic centers

  Research has shown that the proximal conformation facilitates optimal positioning of the [2Fe-2S] cluster of FDX2 relative to the ISCU2 FeS cluster assembly site, enabling efficient electron transfer . The distal-to-proximal transition appears to be influenced by the C-terminal region, particularly residue Asn175.

• How does the redox potential of FDX2 influence its electron transfer specificity in FeS cluster assembly?

  The redox potential of FDX2 is a critical determinant of its electron transfer capabilities and functional specificity:

  Thermodynamic Considerations:

  • The midpoint redox potential (E°') determines the thermodynamic driving force for electron transfer

  • For efficient electron transfer, the potential of FDX2 must be sufficiently negative to reduce its target in the ISC machinery

  • Related ferredoxins typically have negative redox potentials; for example, Rhodobacter capsulatus FdV has E°' = -220 mV at pH 7.5

  Functional Implications:

  • Too high (less negative) redox potential can limit electron donation capacity

  • Too low (more negative) potential might lead to non-specific electron transfer

  • The specific redox potential of FDX2 likely evolved to match precisely the requirements of the ISC machinery

  Specificity Determinants:

  • The distinct redox properties of FDX1 and FDX2 contribute to their specificity for different pathways:

    • FDX1: Optimized for electron transfer to cytochromes P450 in steroidogenesis

    • FDX2: Optimized for electron transfer in Fe/S cluster biosynthesis

  Experimental Approaches:

  • Protein engineering to alter redox potential through targeted mutations

  • Correlation of redox potential changes with functional outcomes in FeS cluster assembly

  • Spectroelectrochemical measurements to determine precise potential values in different protein contexts

  Physiological Relevance:

  • The matched redox properties between FDX2 and its electron acceptor in the ISC machinery ensure efficient and specific electron transfer

  • Environmental factors (pH, ionic strength) may modulate the effective redox potential in vivo

  • Redox potential differences between FDX2 and alternate electron donors explain their varying efficiencies in supporting FeS cluster assembly

  The precisely tuned redox potential of FDX2 represents an important mechanism for ensuring the specificity of electron flow in mitochondrial FeS cluster biogenesis.

• What are the current methodological challenges in distinguishing FDX2's roles in [2Fe-2S] versus [4Fe-4S] cluster assembly?

  Distinguishing FDX2's potentially distinct roles in [2Fe-2S] versus [4Fe-4S] cluster assembly presents several methodological challenges:

  Spectroscopic Differentiation:

  • [2Fe-2S] and [4Fe-4S] clusters have overlapping spectroscopic features

  • Multiple complementary spectroscopic methods must be employed:

    • UV-visible spectroscopy

    • CD spectroscopy

    • EPR spectroscopy

    • Mössbauer spectroscopy for detailed iron site characterization

  Sequential Assembly Complexity:

  • [4Fe-4S] clusters often form via [2Fe-2S] cluster intermediates

  • Distinguishing direct effects on [4Fe-4S] assembly from indirect effects via [2Fe-2S] assembly is challenging

  • Temporal resolution in experimental design is critical

  Protein-Specific Readouts:

  • Using target proteins that specifically require either cluster type:

    • Ferredoxins (containing [2Fe-2S] clusters)

    • Aconitase or lipoate synthase (containing [4Fe-4S] clusters)

    • Monitoring assembly on these specific targets can help differentiate roles

  Isolation of Assembly Steps:

  • Development of in vitro systems that can separate [2Fe-2S] and [4Fe-4S] assembly steps

  • Selective inhibition of specific components in the [2Fe-2S] to [4Fe-4S] conversion pathway

  • Reconstitution approaches with defined components

  Genetic and Cellular Approaches:

  • Conditional depletion systems for FDX2 with time-course analysis

  • Complementation with FDX2 variants that might differentially affect [2Fe-2S] versus [4Fe-4S] assembly

  • Combined depletion of FDX2 with other factors specific to either pathway

  Addressing these challenges requires innovative experimental design, complementary methodologies, and careful interpretation of results to build a comprehensive understanding of FDX2's potentially distinct roles in assembling different types of FeS clusters.