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Rationally tuning the reduction potential of a single cupredoxin beyond the natural range

Nature volume 462, pages 113116 (05 November 2009) | Download Citation


Redox processes are at the heart of numerous functions in chemistry and biology, from long-range electron transfer in photosynthesis and respiration to catalysis in industrial and fuel cell research. These functions are accomplished in nature by only a limited number of redox-active agents. A long-standing issue in these fields is how redox potentials are fine-tuned over a broad range with little change to the redox-active site or electron-transfer properties. Resolving this issue will not only advance our fundamental understanding of the roles of long-range, non-covalent interactions in redox processes, but also allow for design of redox-active proteins having tailor-made redox potentials for applications such as artificial photosynthetic centres1,2 or fuel cell catalysts3 for energy conversion. Here we show that two important secondary coordination sphere interactions, hydrophobicity and hydrogen-bonding, are capable of tuning the reduction potential of the cupredoxin azurin over a 700 mV range, surpassing the highest and lowest reduction potentials reported for any mononuclear cupredoxin, without perturbing the metal binding site beyond what is typical for the cupredoxin family of proteins. We also demonstrate that the effects of individual structural features are additive and that redox potential tuning of azurin is now predictable across the full range of cupredoxin potentials.

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This material is based on work supported by the US National Science Foundation under award no. CHE 05-52008. We thank E. Marshall, D. Poe, A. Huang and J. Li for discussions, and for help in protein expression and purification, and in spectroscopic and electrochemical data collection. N.M.M. is an NIH Predoctoral trainee, supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 5 T32 GM070421 from the National Institute of General Medical Sciences.

Author Contributions N.M.M. performed most of the experimentation, led the study and authored most of the manuscript. D.K.G. and T.D.W. contributed intellectually and assisted in experimentation and editing. Crystal diffraction patterns were collected by H.R. and refined into PDB structures by Y.-G.G. M.J.N. assisted with EPR data collection and simulation. Y.L. designed and guided the project, and edited the paper.

Author information


  1. Department of Chemistry, University of Illinois, Urbana-Champaign, Illinois 61801, USA

    • Nicholas M. Marshall
    • , Dewain K. Garner
    • , Tiffany D. Wilson
    • , Yi-Gui Gao
    • , Mark J. Nilges
    •  & Yi Lu
  2. Biology Department, Brookhaven National Laboratory, Upton, New York 11973-5000, USA

    • Howard Robinson


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Corresponding author

Correspondence to Yi Lu.

Crystallographic data have been deposited at the Protein Data Bank with the following PDB codes: N47S/M121L Az, 3JT2; N47S/F114N Az, 3JTB; F114P/M121Q Az, 3IN0.

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    Supplementary Information

    This file contains Supplementary Notes, Supplementary Figures S1-12 with Legends, Supplementary Tables S1-S4 and Supplementary References.

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