Rational design of a structural and functional nitric oxide reductase

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Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally1,2,3, it is more difficult to design functional proteins4,5,6,7,8. In comparison to recent successes in designing non-metalloproteins4,6,7,9,10, it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymes5,8,11,12,13,14,15,16,17,18,19,20. This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem FeB centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico.

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Figure 1: Crystal structure of rationally designed Fe B Mb overlays closely with minimized computer model.
Figure 2: Designed FeBMb has NO reduction activity in the presence of Fe2+.
Figure 3: Product of NO reaction with Fe B Mb is N 2 O.

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Protein Data Bank

Data deposits

Crystallographic data for FeBMb have been deposited in the Protein Data Bank with accession number 3K9Z.


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We thank M. J. Nilges for help with EPR analysis, S. L. Mullen and F. Sun for aiding in GC/MS data collection, E. Lee for help with computational modelling, N. M. Marshall for providing Azurin protein, J. R. Askim for help in FeBMb expression and purification, and T. Hayashi and P. Moënne-Loccoz for suggestions regarding N2O detection in solution. This work was supported by the US National Institutes of Health (GM062211).

Author Contributions N.Y. and Y.-W.L. performed most of the experimentation and wrote most of the manuscript. B.S.R. helped with the initial design of mutants and experiments. X.Z. and L.L. assisted in experimentation. K.D.M. performed computational modelling. Y.-G.G. guided crystallization and refined the crystal structure. H.R. collected crystal diffraction data. Y.L. designed, guided the project and edited the manuscript.

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Correspondence to Yi Lu.

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Yeung, N., Lin, Y., Gao, Y. et al. Rational design of a structural and functional nitric oxide reductase . Nature 462, 1079–1082 (2009) doi:10.1038/nature08620

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