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Atomic resolution crystallography reveals how changes in pH shape the protein microenvironment

Nature Chemical Biology volume 2, pages 259–264 (2006)Cite this article

An Erratum to this article was published on 01 June 2006

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Abstract

Hydrogen atoms are a vital component of enzyme structure and function1,2,3,4. In recent years, atomic resolution crystallography (≥1.2 Å) has been successfully used to investigate the role of the hydrogen atom in enzymatic catalysis5,6,7,8,9. Here, atomic resolution crystallography was used to study the effect of pH on cholesterol oxidase from Streptomyces sp., a flavoenzyme oxidoreductase. Crystallographic observations of the anionic oxidized flavin cofactor at basic pH are consistent with the UV-visible absorption profile of the enzyme and readily explain the reversible pH-dependent loss of oxidation activity. Furthermore, a hydrogen atom, positioned at an unusually short distance from the main chain carbonyl oxygen of Met122 at high pH, was observed, suggesting a previously unknown mechanism of cofactor stabilization. This study shows how a redox active site responds to changes in the enzyme's environment and how these changes are able to influence the mechanism of enzymatic catalysis.

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Figure 1: Electron density at atomic resolution.
Figure 2: Changes of FAD protonation state and multiple populations of hydrogen atoms as a function of pH.
Figure 3: Populations of hydrogen atom positions in the region of the FAD cofactor.
Figure 4: Protonation state of His447 as a function of pH.
Figure 5: A model illustrating the pH-dependent changes in the CO active site, showing FAD interacting with the main chain of Met122 and His447 interacting with the side chains of Asn321 and Asn323.

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  • 18 April 2006

    reference 2 at end of sentence changed to superscript 2

Notes

  1. *Note: In the version of this article initially published, the seventh sentence of the fifth paragraph is incorrect. It should read “Hydrogen atoms for the side chains were also clearly visible when the residues showed low temperature factors (typically below 8 Å2).” The error has been corrected in the HTML and PDF versions of the article.

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Acknowledgements

The authors thank N. Sampson for providing pure protein material for these studies and for numerous useful discussions; W. Scott, K. Karplus, G. Petsko and B. Yazar for useful discussions; T. Swartz and R. Chan for the generous loan of spectrophotometric equipment and help with data processing; and G. Gadda and M. Ghanem for their help with the interpretation of the spectroscopic results.

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Authors and Affiliations

  1. Department of Molecular, Cell and Developmental Biology, 1156 High Street, University of California, Santa Cruz, 95064, California, USA

    Artem Y Lyubimov

  2. Department of Chemistry and Biochemistry, 1156 High Street, University of California, Santa Cruz, 95064, California, USA

    Paula I Lario, Ibrahim Moustafa & Alice Vrielink

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  1. Artem Y Lyubimov
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Correspondence to Alice Vrielink.

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Supplementary information

Supplementary Fig. 1

Spectroscopic evidence of FAD deprotonation. (PDF 315 kb)

Supplementary Fig. 2

Variation of geometric parameters of the active site His447 vs. pH. (PDF 136 kb)

Supplementary Table 1

Data collection and structure refinement statistics. (PDF 54 kb)

Supplementary Table 2

Predicted pKa values for histidine sidechains. (PDF 50 kb)

Supplementary Methods (PDF 69 kb)

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Lyubimov, A., Lario, P., Moustafa, I. et al. Atomic resolution crystallography reveals how changes in pH shape the protein microenvironment. Nat Chem Biol 2, 259–264 (2006). https://doi.org/10.1038/nchembio784

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