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Nitrite reductase activity within an antiparallel de novo scaffold

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Abstract

Copper nitrite reductase (CuNiR) is a copper enzyme that converts nitrite to nitric oxide and is an important part of the global nitrogen cycle in bacteria. The relatively simple CuHis3 binding site of the CuNiR active site has made it an enticing target for small molecule modeling and de novo protein design studies. We have previously reported symmetric CuNiR models within parallel three stranded coiled coil systems, with activities that span a range of three orders of magnitude. In this report, we investigate the same CuHis3 binding site within an antiparallel three helical bundle scaffold, which allows the design of asymmetric constructs. We determine that a simple CuHis3 binding site can be designed within this scaffold with enhanced activity relative to the comparable construct in parallel coiled coils. Incorporating more complex designs or repositioning this binding site can decrease this activity as much as 15 times. Comparing these constructs, we reaffirm a previous result in which a blue shift in the 1s to 4p transition energy determined by Cu(I) X-ray absorption spectroscopy is correlated with an enhanced activity within imidazole-based constructs. With this step and recent successful electron transfer site designs within this scaffold, we are one step closer to a fully functional de novo designed nitrite reductase.

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Acknowledgements

V.L.P thanks the National Institutes of Health for financial support of this research (GM141086). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. A.G.T. acknowledges support from the Rackham Merit Fellowship (University of Michigan), as well as training grant support from the University of Michigan Chemistry–Biology Interface (CBI) training program (NIH grant 5T32GM008597) and support from the Chateaubriand Fellowship (Embassy of France in the United States).

Funding

V.L.P thanks the National Institutes of Health for financial support of this research (GM141086). Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research, and by the National Institutes of Health, National Institute of General Medical Sciences (including P41GM103393). The contents of this publication are solely the responsibility of the authors and do not necessarily represent the official views of NIGMS or NIH. A.G.T. acknowledges support from the Rackham Merit Fellowship (University of Michigan), as well as training grant support from the University of Michigan Chemistry-Biology Interface (CBI) training program (NIH grant 5T32GM008597) and support from the Chateaubriand Fellowship (Embassy of France in the United States).

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Authors and Affiliations

  1. Department of Chemistry, University of Michigan, Ann Arbor, MI, USA

    Karl J. Koebke, Elizabeth C. Manickas, Aniruddha Deb, James E. Penner-Hahn & Vincent L. Pecoraro

  2. Program in Chemical Biology, University of Michigan, Ann Arbor, MI, USA

    Alison G. Tebo

  3. Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA

    Alison G. Tebo

  4. Department of Biophysics, University of Michigan, Ann Arbor, MI, USA

    James E. Penner-Hahn

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  1. Karl J. Koebke
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  2. Alison G. Tebo
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  3. Elizabeth C. Manickas
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  5. James E. Penner-Hahn
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  6. Vincent L. Pecoraro
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Correspondence to Vincent L. Pecoraro.

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The datasets generated during and analyzed during the current study are available from the corresponding author on reasonable request.

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Supporting information includes a table of Cu(I) EXAFS fitting parameters and figures of the best Cu(I) EXAFS fit for each construct reported.

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Koebke, K.J., Tebo, A.G., Manickas, E.C. et al. Nitrite reductase activity within an antiparallel de novo scaffold. J Biol Inorg Chem 26, 855–862 (2021). https://doi.org/10.1007/s00775-021-01889-1

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