Protonation/Deprotonation of a Conserved Aspartate Residue Determines the Redox Potential of Low-Potential Ferredoxin

Kernkonzepte

The protonation state of a conserved aspartate residue (Asp64 in Bacillus thermoproteolyticus ferredoxin) acts as a switch, controlling the redox potential and electron transfer in low-potential [4Fe-4S] and [3Fe-4S] ferredoxins.

Zusammenfassung

• Bibliographic Information: Not applicable - the provided content does not include bibliographic information.
• Research Objective: This research investigates the factors controlling the redox potential of low-potential ferredoxins, specifically focusing on the role of hydrogen bonding networks around the [4Fe-4S] cluster.
• Methodology: The researchers employed neutron crystallography to determine the precise hydrogen-bonding network in Bacillus thermoproteolyticus ferredoxin (BtFd). They then used density functional theory (DFT) calculations to analyze the effect of protonation states on the redox stability of the [4Fe-4S] cluster. Additionally, site-directed mutagenesis and air-oxidation kinetics experiments were conducted to validate the computational findings.
• Key Findings:
  • Neutron structure analysis of BtFd revealed the detailed hydrogen-bonding network surrounding the [4Fe-4S] cluster.
  • DFT calculations demonstrated that the protonation state of Asp64, a residue near the [4Fe-4S] cluster, significantly influences the cluster's redox potential. Protonation stabilizes the reduced state.
  • Mutating Asp64 to neutral or positively charged residues slowed the air-oxidation rate of the [4Fe-4S] cluster, confirming the importance of its protonation state.
  • Similar effects of analogous aspartate/glutamate residues were observed in ferredoxins from other organisms, suggesting a conserved mechanism.
• Main Conclusions: The protonation state of a conserved aspartate or glutamate residue near the [4Fe-4S]/[3Fe-4S] cluster acts as a switch, controlling the redox potential and electron transfer in low-potential ferredoxins. This mechanism likely prevents electron backflow and ensures efficient electron transfer to partner proteins.
• Significance: This study provides the first identification of an intrinsic factor controlling the redox potential in low-potential ferredoxins, advancing our understanding of electron transfer regulation in biological systems.
• Limitations and Future Research: Further research is needed to determine the complex structures of ferredoxin with its electron donor and acceptor partners. This would provide a more comprehensive understanding of the dynamic interplay between protonation states and electron transfer in these systems.

Statistiken

The redox potential of wild-type BtFd was estimated to be -346 mV.
The D64A and D64N mutations shifted the redox potential to -276 mV and -286 mV, respectively.
The D64E mutation did not significantly affect the redox potential (-346 mV).
Wild-type BtFd exhibited a T1/2 of 100 seconds for air oxidation.
The D64A and D64N mutations resulted in a T1/2 of 166 and 170 seconds, respectively.
The pKa of Asp64 in oxidized BtFd was estimated to be approximately 4.9.
The pKa of Asp64 in reduced BtFd is estimated to be between pH 6.0 and 7.0 based on air-oxidation rate measurements.

Zitate

"These findings provide the first identification of intrinsic factors controlling the redox stability of the [4Fe-4S] cluster in low-potential ferredoxins."
"These results suggest that the protonation state of the sidechain of Asp64 plays important roles for the stability of the reduced state, and furthermore in the control of electron transfer."
"To our knowledge, this is the first proposed mechanism of the intrinsic control factor of redox potentials of the [4Fe-4S]/[3Fe-4S] cluster in low-potential ferredoxins."

Wichtige Erkenntnisse aus

Protonation/deprotonation-driven switch for the redox stability of low-potential [4Fe-4S] ferredoxin

by Wada,K., Kob... um www.biorxiv.org 09-14-2024

https://www.biorxiv.org/content/10.1101/2024.09.12.612615v2

Tiefere Fragen

How might the protonation-dependent redox switch in ferredoxin be influenced by changes in the cellular environment, such as pH fluctuations or interactions with other molecules?

The protonation-dependent redox switch in ferredoxin, primarily governed by the conserved aspartate/glutamate residue near the [4Fe-4S] or [3Fe-4S] cluster, is inherently sensitive to changes in the cellular environment. Here's how:

pH fluctuations: The protonation state of the Asp/Glu residue is directly dependent on the surrounding pH. As pH decreases (becomes more acidic), protonation of the Asp/Glu sidechain becomes more favorable. This protonation stabilizes the reduced state of the Fe-S cluster, shifting the redox potential more positive and potentially slowing down electron transfer. Conversely, an increase in pH (more alkaline) promotes deprotonation, favoring the oxidized state and facilitating electron acceptance. Therefore, fluctuations in cellular pH, such as those occurring in response to metabolic processes or environmental stress, can dynamically regulate ferredoxin's redox potential and electron transfer activity.

Interactions with other molecules:

Electrostatic interactions: The presence of charged molecules, such as ions or other proteins, in the vicinity of the Asp/Glu residue can influence its pKa and consequently its protonation state. For instance, a high concentration of positive charges near the Asp/Glu could stabilize its deprotonated form, even at a lower pH.
Hydrogen bonding: Formation of hydrogen bonds with the Asp/Glu residue can alter its proton affinity. Molecules capable of donating hydrogen bonds could stabilize the deprotonated form, while those accepting hydrogen bonds could favor protonation.
Protein-protein interactions: Binding of ferredoxin to its redox partners could induce conformational changes that directly impact the Asp/Glu residue. This interaction might alter its solvent accessibility, hydrogen-bonding network, or proximity to charged residues, ultimately influencing its protonation state and the redox potential of the Fe-S cluster.
In essence, the protonation-dependent redox switch in ferredoxin acts as a sensitive sensor of the cellular environment. Fluctuations in pH, ion concentrations, and interactions with other molecules can fine-tune its redox potential, allowing for dynamic regulation of electron transfer processes in response to changing cellular conditions.

Could other residues, beyond the conserved aspartate/glutamate, play a secondary role in modulating the redox potential of ferredoxin?

While the conserved aspartate/glutamate residue plays a primary role in the protonation-dependent redox switch of ferredoxin, other residues can indeed contribute to fine-tuning its redox potential. These secondary influences can arise from:

Hydrogen bonding networks: Residues capable of forming hydrogen bonds with the sulfur atoms of the Fe-S cluster or with the primary Asp/Glu residue can influence the redox potential. For example, the study highlighted the importance of accurately mapping the hydrogen-bonding network around the Fe-S cluster, demonstrating that even a single hydrogen bond can significantly impact the redox properties. Mutations or structural changes affecting these secondary hydrogen bonds could subtly adjust the redox potential.

Electrostatic interactions: Polar or charged residues near the Fe-S cluster can create an electrostatic environment that influences electron affinity. Positively charged residues would stabilize the reduced state (more negative), while negatively charged residues would favor the oxidized state. Even distant residues can contribute to the overall electrostatic field around the active site.

Hydrophobic environment: The degree of solvent exposure of the Fe-S cluster can also impact its redox potential. Hydrophobic residues surrounding the cluster can shield it from water molecules, influencing its reduction potential. Changes in the hydrophobicity of the surrounding environment, perhaps due to mutations or conformational changes, could fine-tune the redox properties.

Conformational changes: Dynamic motions within the protein structure, even those distant from the active site, can propagate to the Fe-S cluster and alter its environment. These changes might affect hydrogen bonding networks, electrostatic interactions, or solvent exposure, ultimately influencing the redox potential.
Therefore, while the conserved Asp/Glu acts as a primary redox switch, a network of secondary interactions involving other residues, the protein's structure, and the surrounding solvent environment can fine-tune the redox potential of ferredoxin. This intricate interplay allows for precise control of electron transfer rates and fine-tuning of ferredoxin's function in response to diverse cellular needs.

What are the broader implications of understanding and potentially manipulating redox potentials in biological systems, particularly in the context of synthetic biology or bioengineering applications?

Understanding and manipulating redox potentials in biological systems, particularly those involving electron transfer proteins like ferredoxin, holds immense potential for various biotechnological and bioengineering applications:

Synthetic biology:

Designing artificial metabolic pathways: By manipulating the redox potentials of electron carriers like ferredoxin, synthetic biologists can control the flow of electrons in engineered metabolic pathways. This control enables the optimization of existing pathways or the creation of entirely new ones for producing biofuels, pharmaceuticals, or other valuable compounds.
Building biosensors: Redox-active proteins can be engineered to function as biosensors by coupling their redox potential to the presence of specific analytes. Changes in the redox state, detectable through electrochemical or optical means, can then be used to quantify the target molecule.

Bioengineering:

Improving bioremediation: Manipulating the redox potential of enzymes involved in bioremediation processes can enhance their efficiency in degrading pollutants. For example, optimizing electron transfer chains in bacteria used for cleaning up oil spills or degrading toxic waste can significantly improve their effectiveness.
Developing bioelectronics: Redox-active proteins can be integrated into bioelectronic devices, such as biofuel cells or biosensors. By controlling and interfacing with their redox potentials, these devices can harness biological processes for energy generation or sensing applications.

Biocatalysis:

Optimizing enzymatic reactions: Many industrial processes rely on enzymes that utilize redox reactions. Understanding and manipulating the redox potentials of these enzymes can lead to improved catalytic efficiency, altered substrate specificity, or enhanced stability, ultimately leading to more efficient and sustainable biocatalytic processes.

Therapeutic applications:

Targeting redox imbalances: Many diseases, including cancer and neurodegenerative disorders, are associated with redox imbalances. Manipulating the redox potential of specific proteins could provide new therapeutic strategies for restoring redox homeostasis and treating these conditions.
Overall, the ability to understand and manipulate redox potentials in biological systems opens up exciting possibilities in synthetic biology, bioengineering, and medicine. By fine-tuning the electron transfer properties of proteins like ferredoxin, we can design new biological systems with tailored functionalities, leading to advancements in sustainable energy production, environmental remediation, biosensing, biocatalysis, and disease treatment.