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Conformational changes in an ultrafast light-driven enzyme determine catalytic activity

Nature volume 456, pages 1001–1004 (2008)Cite this article

Abstract

The role of conformational changes in explaining the huge catalytic power of enzymes is currently one of the most challenging questions in biology1,2,3,4,5,6,7. Although it is now widely regarded that enzymes modulate reaction rates by means of short- and long-range protein motions3,4,5,6,7, it is almost impossible to distinguish between conformational changes and catalysis. We have solved this problem using the chlorophyll biosynthetic enzyme NADPH:protochlorophyllide (Pchlide) oxidoreductase, which catalyses a unique light-driven reaction involving hydride and proton transfers8. Here we report that prior excitation of the enzyme-substrate complex with a laser pulse induces a more favourable conformation of the active site, enabling the coupled hydride and proton transfer reactions to occur. This effect, which is triggered during the Pchlide excited-state lifetime and persists on a long timescale, switches the enzyme into an active state characterized by a high rate and quantum yield of formation of a catalytic intermediate. The corresponding spectral changes in the mid-infrared following the absorption of one photon reveal significant conformational changes in the enzyme, illustrating the importance of flexibility and dynamics in the structure of enzymes for their function.

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Figure 1: Homology model of POR from Synechocystis24.
Figure 2: Evolution-associated difference spectra resulting from a global analysis, as a function of illumination time.
Figure 3: Target analysis 26 of the illumination-dependent POR pump–probe data sets.
Figure 4: Mid-infrared absorption difference data.

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Acknowledgements

This research was supported by The Netherlands Organization for Scientific Research through the Dutch Foundation for Earth and Life Sciences (Investment Grant no. 834.01.002). M.L.G. is grateful to NWO-ALW for providing financial support with a long-term fellowship (Grant no. 831.00.004) and O.S. received support from NWO-CW (Grant no. 700.53.307). D.J.H. and C.N.H. gratefully acknowledge support from the Biotechnology and Biological Sciences Research Council, UK. We thank H. Fidder and F. van Mourik for reading the manuscript.

Author Contributions O.A.S., D.J.H., M.T.A. and M.L.G. produced the samples and performed all of the experiments. O.A.S., I.H.M.v.S. and M.L.G. analysed the data. D.J.H., C.N.H., R.v.G. and M.L.G. coordinated the study, designed the experiments and wrote the paper. All authors discussed the results and commented on the manuscript.

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Authors and Affiliations

  1. Department of Physics and Astronomy, Faculty of Sciences, Vrije Universiteit, De Boelelaan 1081, 1081 HV Amsterdam, The Netherlands,

    Olga A. Sytina, Maxime T. Alexandre, Ivo H. M. van Stokkum, Rienk van Grondelle & Marie Louise Groot

  2. Manchester Interdisciplinary Biocentre, University of Manchester, 131 Princess Street, Manchester M1 7DN, UK ,

    Derren J. Heyes

  3. Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK,

    C. Neil Hunter

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  1. Olga A. Sytina
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Correspondence to C. Neil Hunter.

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Sytina, O., Heyes, D., Hunter, C. et al. Conformational changes in an ultrafast light-driven enzyme determine catalytic activity. Nature 456, 1001–1004 (2008). https://doi.org/10.1038/nature07354

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