Redox Potential Tuning
Figure 1
Figure 2
We have striven to tune the redox potentials (Eº) of metal centers in a single protein to cover the entire range of biologically relevant Eº from -1 to +1 V. Redox potential is at the heart of many chemical and biological processes from electron transfer (ET) in photosynthesis and respiration to catalysis in water oxidation and N2 fixation. Since the primary coordination spheres defines ET efficiency and catalytic reactivity of metalloproteins, the reactivity can only be tuned by altering secondary coordination sphere interactions to ensure a minimal effect on ET or catalytic efficiency. While it is well known that nature uses a limited set of primary coordination spheres to span a wide range of Eº, the factors that control the Eº, especially those that do not perturb the primary coordination spheres, remain elusive.

To address this issue, we explore novel concepts through systematically tuning the Eº of metal centers in our protein systems by:
  1. rational design of the secondary coordination sphere interactions around the primary coordination spheres
  2. incorporation of unnatural amino acids, and
  3. directed protein evolution.

We have demonstrated redox potential tuning of a single cupredoxin, azurin, across the entire 2V natural range, which is broader than that of native cupredoxins and their mutants (~500 mV), through fine-tuning of three non-covalent secondary coordination sphere interactions: hydrophobicity, hydrogen-bonding, and peptide bond oxygen interactions (1). We have further demonstrated that these features are additive, making redox potential tuning of azurin predictable and unprecedented, while introducing a new level of understanding of long-range, non-covalent interactions in tuning protein functions. The same strategy has also been applied in tuning the redox potentials of a dinuclar CuA system, indicating that the effects are generalizable (2). Using a series of azurin mutants with tuned redox potentials, we have also succeeded in lowering the reorganization energy of ET below that of the native protein, resulting in faster electron transfer rates (3).
Using the knowledge gained from this study we demonstrated the observation of a Marcus inverted region of electron transfer in a non-derivatized protein system (4, 5).

These projects will allow us to advance fundamental knowledge of structural features for systematic control the metal centers’ Eº without perturbing their primary coordination spheres and to demonstrate transformative potentials of such research in applying the knowledge to design:
  1. water-soluble and stable redox agents with tunable Eº for biochemical studies
  2. ET proteins with controlled Eº that can transfer electrons in the inverted region of Marcus theory for applications in photosynthesis and respiration, and
  3. biocatalysts with tunable activities through potential tuning.


References
  1. 1. Hosseinzadeh, P. et al. Proc. Natl. Acad. Scie. 113, (2) 262-267.
  2. 2. New, S. Y., et al. Chem. Commun. 2012, 48,(35) 4217-4219.
  3. 3. Farver, O., et al. Proceedings of the National Academy of Sciences 2013, 110, 10536-10540.
  4. 4. Farver, O., et al. The Journal of Physcal Chemstry Letters 2015 6, 100-105.
  5. 5. Parisa Hosseinzadeh and Yi Lu, Biochem. Biophys. Acta (BBA) - Bioenergetics, 2015.